CN101121938B - Method for preparing foot-and-mouth disease antigen - Google Patents

Method for preparing foot-and-mouth disease antigen Download PDF

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CN101121938B
CN101121938B CN2007100900359A CN200710090035A CN101121938B CN 101121938 B CN101121938 B CN 101121938B CN 2007100900359 A CN2007100900359 A CN 2007100900359A CN 200710090035 A CN200710090035 A CN 200710090035A CN 101121938 B CN101121938 B CN 101121938B
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CN101121938A (en
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柳纪省
张志芳
李志勇
易咏竹
殷相平
白银梅
李轶女
李宝玉
李学瑞
杨彬
兰喜
沈桂芳
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Biotechnology Research Institute of CAAS
Lanzhou Veterinary Research Institute of CAAS
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Biotechnology Research Institute of CAAS
Lanzhou Veterinary Research Institute of CAAS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/125—Picornaviridae, e.g. calicivirus
    • A61K39/135—Foot- and mouth-disease virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/085—Picornaviridae, e.g. coxsackie virus, echovirus, enterovirus
    • C07K14/09—Foot-and-mouth disease virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/866—Baculoviral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Abstract

The invention provides a method to express foot-and-mouth disease antigen by using a recombinant baculorirus in the body of an insect. Different genomes of foot-and-mouth disease are respectively cloned into the baculovirus vector to achieve a transfer vector; the constructed transfer vector transfection baculovirus is used for DNA recombination to achieve a recombinant baculovirus; an insect host is infected by the recombinant baculovirus; the infected insect host is cultivated to achieve expression of foot-and-mouth disease antigen; and finally, the expressed foot-and-mouth disease antigen is collected and purified. The invention adopts baculovirus expression system to produce foot-and-mouth disease antigen in a silkworm bioreactor safely and efficiently, and the prepared antigen by the method has high safety, and vaccines can be produced directly for animal immunity. The method for preparation of foot-and-mouth disease antigen does not need any plant, has no waste discharge and consumes little power, water or other resources; the production cost is relatively low compared with traditional preparation methods; besides, the method has the advantages of high security and efficiency, low energy-consumption, low cost, etc.

Description

A kind of preparation method of foot-and-mouth disease antigen
Technical field
The present invention relates to the antigenic method of a kind of preparation, relate in particular to a kind of method of utilizing recombinant baculovirus at insect expression in vivo foot-and-mouth disease antigen, belong to the genetically engineered field.
Background technology
Foot and mouth disease be by foot and mouth disease virus (Foot-and-mouth disease virus FMDV) causes a kind of acute, height contact, infectious fever artiodactylous, with propagate rapidly, infection rate is high and celebrated.This disease will be in case outburst will cause enormous economic loss to morbidity state, and countries in the world are very paid attention to the research of this disease, and World Organization for Animal Health classifies it first of category-A transmissible disease as.Foot and mouth disease belongs to the member of Picornaviridae Hostis, and A, O, C, Asia I, SA T1, SA T2 and 7 serotypes of SA T3 type are arranged.The anti-system of China's foot and mouth disease is still to put prevention first at present.Although traditional vaccine is still occupied an leading position in the foot and mouth disease prevention and control, the production cost costliness, duration of immunity is short, and Hazard Factor such as viral escape are difficult to avoid in the vaccine production process.Therefore, traditional vaccine is needed badly and is improved, and development safely, the foot and mouth disease molecular vaccine still seems very necessary efficiently.
Along with development of high-tech such as biotechnology, genetically engineereds, people recognize by engineered means, utilize bio-reactor to efficiently express the foot and mouth disease gene, be expected to reach purpose (the Conneely O.M. that increases substantially foot-and-mouth disease antigen output, reduces production costs, Biotechnology in the Feed Industry, T.P.Lyons (Ed), Alltech Technical Publications.Nicholasville, K Y.57-66,1992).
This bio-reactor of baculovirus expression system is that the eighties is set up.(Smith since nineteen eighty-three utilizes baculovirus expression system to efficiently express people's alpha-interferon first, Mol.Cell Biol., 3:2156-2165,1983), existing dozens of foreign gene has obtained efficiently expressing, only just there are alpha-interferon (Yang Guanzhen etc. in China, Acta Biochimica et Biophysica Sinica, 22:355-361,1990), arrowhead proteinase inhibitor (season equality, silkworm industry science, 21:223-227,1995), Mareks disease virus Glycoprotein B (Xiao Qingli etc., silkworm industry science, 23:104-108,1997) etc. multiple.The advantage of utilizing this system to produce foot-and-mouth disease antigen is: 1. the expression efficiency of this expression system is high, and output can reach the level of 10 milligrams of level/worms.Thereby can reduce the production cost of foot-and-mouth disease antigen greatly and make and become possibility by gene engineering method scale operation foot-and-mouth disease antigen; 2. this expression system is an eukaryotic expression system, the exogenous protein of its expression can carry out posttranslational modification, make it similar to natural product with biological activity etc. at biochemical property, this has normal protein structure for expressed foot-and-mouth disease antigen and the biology immunocompetence provides assurance.At present, baculovirus expression system, silkworm especially wherein (Bm)-silkworm baculovirus (BmNPV) expression system are one of individual expression systems of eukaryote that has most in the world business development value.
Summary of the invention
Technical problem to be solved by this invention is to overcome the deficiencies in the prior art, and a kind of method of utilizing baculovirus expression system to express foot-and-mouth disease antigen safely, efficiently in insect body is provided.
Technical problem to be solved by this invention is achieved through the following technical solutions:
A kind of method for preparing foot-and-mouth disease antigen comprises: different genomes of foot-and-mouth disease is cloned into respectively in the baculovirus delivery carrier, obtains transfer vector; Carry out the DNA reorganization with the transfer vector transfection baculovirus that is obtained, obtain recombinant baculovirus; Use the recombinate shape virus infection insect host; Cultivating infected insect host makes it carry out the foot-and-mouth disease antigen expression; Results and the expressed antigen of purifying.
Wherein, described different genomes of foot-and-mouth disease is preferably from the base sequence shown in SEQ ID NO:1, SEQ ID NO:3 or the SEQ ID NO:5;
Described baculovirus delivers carrier preferably from AcRP23-lacZ, AcRP6-SC, AcUW1-lacZ, BacPAK6, Bac to Pac, Bacmid, BlucBacII (pETL), p2Bac, p2Blue, p89B310, pAc360, pAc373, pAcAB3, pAcAB 4, PAcAS3, pAcC129, pAcC4, DZI, pAcGP67, pAcIE1, pAcJP1, pAcMLF2, pAcMLF 7, pAcMLF 8, pAcMP1, pAcMP2, pAcRP23, pAcRP 25, pAcRW4, pAcsMAG, pAcUW1, pAcUW21, pAcUW2A, pAcUW2B, pAcUW3, pAcUW31, pAcUW41, pAcUW42, pAcUW43, pAcUW51, pAcVC2, pAcVC 3, pAcYM1, pAcJcC5, pBac1, pBac2, pBlueBacIII, pBlueBacHis, pEV55, mXIV, pIEINeo, pJVETL, pJVNhel, pJVP10, pJVrsMAG, pMBac, pP10, pPAK1, pPBac, pSHONEX 1.1, pSYN XIV VI+, pSYNVI+wp, pSYNXIV VI-, pVL1391, pVL 1392, pVL 1393, pVL941, pVL 945, pVL 985, pVTBac, pBM030, pUAC-5 or other similar baculovirus homologous recombination or transposon vector, more preferably pVL1393.
Constructed transfer vector is preferably pVL1393 (P1-2A3C), pVL1393 (ORF) or pVL1393 (VP1).
Described baculovirus is selected from BmNPV, AcMNPV, ApNPV, BsSNPV, CfMNPV, EoSNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SlMNPV, SeMNPV or TeNPV, is preferably silkworm baculovirus Bm-NPV-ZJ8;
Described recombinant baculovirus be preferably following any one: (1) Bombyx mori nuclear polyhydrosis virus rBmNPV (ORF), its microbial preservation number is: CGMCC NO.1980; The preservation time is: on March 20th, 2007; Depositary institution is: China Committee for Culture Collection of Microorganisms common micro-organisms center; The preservation address is: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica; (2) Bombyx mori nuclear polyhydrosis virus rBmNPV (P1-2A3C), its microbial preservation number is: CGMCC NO.1979; Depositary institution is: China Committee for Culture Collection of Microorganisms common micro-organisms center; The preservation address is: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica; (3) Bombyx mori nuclear polyhydrosis virus rBmNPV (VP1), its microbial preservation number is: CGMCC NO.1975; Depositary institution is: China Committee for Culture Collection of Microorganisms common micro-organisms center; The preservation address is: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica.
Described insect host is selected from and comprises silkworm (Bombyx mori), wild silkworm (Bombyx mandarina), Semen Ricini silkworm (Philosamia cynthia ricim), wild silkworm (Dictyoploca japanica), Philosamia cynthia (Philosamiacynthia pryeri), tussah (Antheraea pernyi), yamama (Antheraea yamamai), wild giant silkworm (Antheraea polyphymus), autographa california (Atographa califorica), tea geometrid (Ectropisobliqua), cabbage army worm (Mamestra brassicae), prodenia litura (Spodoptera littoralis), autumn mythimna separata (Spodoptera frugiperda), cabbage looper (Trichoplusia ni), armyworm (Thaumetopoeawilkinsoni), bollworm (Heliothis armigera), U.S. bollworm (Heliothis zea), oriental tobacco budworm (Heliothis assulta), cigarette beetle (Heliothis virescens), oriental armyworm (Pseudaletia separata), gypsymoth (Lymantria dispar) etc.; Silkworm (Bombyx mori) more preferably.
Described infection be meant recombinant baculovirus through port food or see through epidermis infect the insect larvae in 1-5 age or pupal cell (more preferably: with the silkworm larva or the pupa in recombinant silkworm baculovirus infection bombyx mori cell or percutaneous puncture-inoculation 1-5 age, after infecting 3-6 days, collect contain the silkworm larva of foot-and-mouth disease antigen or the body fluid or the tissue homogenate of pupa); Wherein, described pupal cell is 1-2 days an early stage tender pupa.
The present invention adopts gene recombination technology, with deriving from the different genes combination of foot and mouth disease virus, comprises P1-2A3C, ORF, VP1 is building up to various baculovirus delivery carriers (as AcRP23-lacZ, AcRP6-SC, AcUW1-lacZ, BacPAK6, Bac to Pac, Bacmid, BlueBacII (pETL), p2Bac, p2Blue, p89B310, pAc360,373, pAcAB3,4, pAcAS3, pAcC129, C4, DZ1, pAcGP67, pAcIE1, pAcJP1, pAcMLF2,7,8, pAcMP1,2, pAcRP23,25, pAcRW4, pAcsMAG, pAcUW1,21,2A, 2B, 3,31,41,42,43,51, pAcVC2,3, pAcYM1, pAcJcC5, pBac1,2, pBlueBacIII, pBlueBacHis, pEV55, mXIV, pIEINeo, pJVETL, pJVNhe1, pJVP10, pJVrsMAG, pMBac, pP10, pPAK1, pPBac, pSHONEX 1.1, pSYN XIV VI+, pSYNVI+wp, pSYNXIVVI-, pVL1391,1392,1393, pVL941,945,985, pVTBac, pBM030, pUAC-5) on, make the different assortment of genes of foot and mouth disease virus, comprise ORF, P1-2A3C, the VP1 gene is in the polyhedron promotor, under p10 promotor or other virus and the control of Eukaryotic strong promoter, by in the body or external (in vivo/in vitro) reorganization, the assortment of genes that foot and mouth disease virus is different comprises P1-2A3C, ORF, VP1 is incorporated on the genome of baculovirus, obtains recombinant virus.Insect larvae or the pupal cell (optimal time be 1-2 days early stage tender pupa) of various means through epidermis infection 1-5 age (optimal time was four or five ages) be eaten down or be adopted to recombinant virus can by per os, expresses the production foot-and-mouth disease virus antigen.
A most preferred technical scheme is among the present invention: with SEQ ID NO:1 (P1-2A3C), base sequence shown in SEQ IDNO:3 (ORF) or the SEQ ID NO:5 (VP1) is inserted on the delivery carrier pVL1393, again by recombinating in the body with total length P1-2A3C, ORF, the VP1 transgenosis is to the genome of silkworm baculovirus parent plant Bm-NPV-ZJ8, substitute the Polyhedrin gene on the genome, by plaque select technology and PCR detection technique, obtain to carry the recombinant silkworm baculovirus rBmNPV (ORF) of foot and mouth disease different genes combination, rBmNPV (P1-2A3C), rBmNPV (VP1).With the silkworm larva or the pupa in its infected silkworm clone or percutaneous puncture-inoculation 1-5 age, breed rBmNPV (ORF), rBmNPV (P1-2A3C), rBmNPV (VP1) in a large number.As rBmNPV (ORF), rBmNPV (P1-2A3C), when rBmNPV (VP1) duplicates in the silkworm body, ORF, P1-2A3C, VP1 express under the control of polyhedron gene (polh) promotor, produce foot-and-mouth disease antigen.After infecting 3-6 days (the best is 5 days), collect and contain the silkworm larva of foot-and-mouth disease antigen or the body fluid of pupa (or whole homogenate), every milliliter of silkworm hemolymph can produce the foot-and-mouth disease antigen more than 10 milligrams, after killing infectious cause of disease, through just obtaining safety, foot-and-mouth disease antigen efficiently behind the protein purification, this kind antigen can be used for preparing vaccine.
The inventive method adopts baculovirus expression system to produce foot-and-mouth disease antigen safely, efficiently in silkworm biological reactor, its production cost significantly is lower than traditional preparation foot-and-mouth disease antigen method (for example by cell proliferation virus preparation foot-and-mouth disease antigen), need not investment founds the factory, the no three wastes, electric power and the consumption of water resources equal energy source are few.Because silkworm China Ministry of Health approval so behind the antigen purification that the inventive method is prepared, security is high, can directly be made the vaccine immunity animal for food medicine dual-purpose insect.
In general, the inventive method can reduce the production cost of foot-and-mouth disease antigen significantly, has plurality of advantages such as safety, efficient, less energy consumption, cost are low.
Description of drawings
Fig. 1, the expression of foot and mouth disease virus P1-2A3C in silkworm larva.
Fig. 2, the expression of foot and mouth disease virus P1-2A3C in silkworm pupa.
Fig. 3, the expression of foot and mouth disease virus total length ORF in silkworm larva.
Fig. 4, the expression of foot and mouth disease virus total length ORF in silkworm pupa.
Fig. 5, the expression of foot and mouth disease virus VP1 in silkworm larva.
Fig. 6, the expression of foot and mouth disease virus VP1 in silkworm pupa.
Embodiment
In order further to set forth the present invention, provide a series of embodiment below.These embodiment are illustrative fully, and they only are used for the present invention is specifically described, and not should be understood to limitation of the present invention.
Test materials
1. e. coli strains E.coli TG1 and DH 5 αAvailable from Promega company; Delivery carrier pVL1393 (available from Invitrogen company), bombyx mori cell BmN, Bombyx mori nuclear polyhydrosis virus parent plant Bm-NPV-ZJ8 are preserved by Biological Technology institute, Chinese Academy of Agricultural Sciences; Foot and mouth disease virus is preserved by foot and mouth disease country of Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences reference laboratory; The antigen detecting agent box is prepared by Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences, and cultivated silkworm breed variety JY1 is preserved by Biological Technology institute, Chinese Academy of Agricultural Sciences.
2. enzyme and reagent: restriction enzyme, ligase enzyme are Promega company product.
3. biochemical reagents: IPTG, X-Gal are Promega company product.Lipofectin, low melting point agarose LMP, PCR test kit, T 4Dna ligase, RNA enzyme, Proteinase K, foetal calf serum and other reagent are purchased the company in Invitrogen, and cell culture medium TC-100 purchases the company in Sigma.
4. substratum: the intestinal bacteria substratum be LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0); The bombyx mori cell substratum is TC-100.
5. the experimentation on animals of foot-and-mouth disease virus gene various combination expression product is carried out at Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences's animal isolation laboratory.
The preparation of embodiment 1 foot-and-mouth disease antigen, purifying and animal immune experiment and virus attack protection experiment
1, the clone and the sequential analysis of P1-2A, 3C gene
1.1 the acquisition of goal gene
The design primer, the method by RT-PCR amplifies foot-and-mouth disease virus antigen albumen P1-2A gene and Nonstructural Protein 3C gene.
The amplimer of designed antigen protein P1-2A gene and Nonstructural Protein 3C gene is,
P1-2A upstream: 5 '-ATA GGATCCACCATGGGAGCCGGGCAATCCAGCC-3 ',
BamH I restriction enzyme site
P1-2A downstream: 5 '-CGC GAATTCTGACATGTCCTCCTGCATCTGGTTG-3 '.
EcoR I restriction enzyme site
3C upstream: 5 '-GCG GAATTCAAGAAACCTGTCGCTTTGAAAGT-3 '.
EcoR I restriction enzyme site
3C downstream: 5 '-ATA AGATCTCTACTCGTGGTGTGGTTCGGGAT-3 '
The BglII restriction enzyme site
From the foot and mouth disease virus cell culture fluid, extract total RNA.Use Oligo (dT) 18 primers under the effect of AMV ThermoScript II the total RNA that extracts, 42 ℃ of reverse transcriptions prepare cDNA.CDNA with acquisition is a template, carries out pcr amplification with Auele Specific Primer.
The PCR reaction system is as follows:
Table 1PCR reaction conditions
Admixture Volume
Sterilization ddH 2O 33μl
10×PCR Buffer 5μl
2.5mM dNTP 4μl
25mMMgCl 2 3μl
5 ' primers 1μl
3 ' primers 1μl
Template DNA 2μl
The Taq enzyme 1μl
Total 50μl
PCR reaction process: 94 ℃ of sex change 10 minutes; 94 ℃ 1 minute, 58 ℃ 1 minute, 72 ℃ 8 minutes, totally 30 circulations.Last extension 10 minutes.
1.2.PCR the purifying of product
P1-2A, the 3C gene product of pcr amplification are carried out 1% agarose gel electrophoresis, find to amplify the fragment of about 2.4kb, 0.7kb.Under ultraviolet lamp, cut the gel that contains corresponding DNA fragments, carry out purifying with the Geneclean test kit then with the sterilization scalpel.Method is as follows: cut gel fragment and weigh, put it in the little centrifuge tube of 1.5ml of sterilization, the 6M NaI that adds 3 times of (v/w) volumes, 37 ℃ with after the gel dissolving, adds 10 μ l glass milk (Glass milk), placed 5 minutes under the room temperature behind the mixing, DNA fully is adsorbed on the glass milk, and 12000rpm centrifugal 5 seconds, it is inferior to give a baby a bath on the third day after its birth with New Wash solution again, all precipitation is upspring at every turn, and centrifugal.At last precipitation is dried the back and add 30 μ l, 0.1 * TE Buffer dissolving DNA, go precipitation after centrifugal, get supernatant and do further to analyze.
1.3. enzyme is cut and ligation
Endonuclease reaction: P1-2A behind the purifying analyzes with BamHI and EcoRI double digestion, and the reaction cumulative volume is 50 μ l, the PCR product 10 μ l of purifying wherein, and the corresponding damping fluid 5 μ l of 10 * enzyme, two kinds of enzymes respectively are 1 μ l, sterilized water is supplied volume.37 ℃ of reactions are more than 2 hours.Transferring plasmid pGEM-3Z is made same endonuclease reaction.Reaction finishes the back in 65 ℃ of deactivations 10 minutes.
Ligation: connect cumulative volume 15 μ l, PCR product 8 μ l, carrier 1 μ l, 5 * T4 DNA connects damping fluid 3 μ l, T4 dna ligase 1 μ l, sterilized water is supplied volume, and 12～14 ℃ of connections are spent the night.
Cut 3C, pGEM-3Z with identical method enzyme, and carry out ligation.
1.4. colibacillary genetic transformation
Use 75mM CaCl 2Preparation e. coli tg1 competent cell.Get the connection mixture 5 μ l of preparation in the step 3, be added in the 200 μ l competent cells, mixing gently, ice bath 30 minutes, 42 ℃ of heat shocks 2 minutes placed rapidly 1～2 minute on ice, add and be incubated to 37 ℃ LB substratum 500 μ l, cultivated 1 hour for 37 ℃, get 100～200 μ l and coat on the LB solid medium flat board that contains 100 μ g/ml penbritins (Amp), be inverted overnight incubation for 37 ℃.
1.5. the preparation of plasmid DNA
(1) the single bacterium colony of picking from the LB flat board that transforms is inoculated in 3ml and contains in the LB substratum of 100 μ g/ml Amp 37 ℃ of overnight incubation.
(2) get 1.5ml bacterium liquid in little centrifuge tube, the centrifugal 4min of 3500rpm removes supernatant.
(3) add Solution I 150 μ l, mixing is placed on 15min on ice.
(4) add Solution II 300 μ l, chloroform 150 μ l leave standstill 5min behind the mixing gently.
(5) add Solution III 450 μ l, mixing is placed on 15min on ice.
(6) the centrifugal 10min of 11000g, supernatant move into new pipe.
(7) add Virahol 450 μ l, mixing is placed on 4 ℃ of 15min.
(8) the centrifugal 6min of 11000g removes supernatant.
(9) add TER 250 μ l, mixing is placed on 37 ℃ of 20min.
(10) add PPt Buffer 300～350 μ l, leave standstill 15min behind the mixing.
(11) the centrifugal 6min of 11000g removes supernatant.
(12) add 75% ethanol, 400 μ l.
(13) the centrifugal 3min of 11000g outwells ethanol, drains the back and adds 0.1 * TE Buffer, 40 μ l dissolving, in-20 ℃ of preservations.
1.6. the evaluation of recon
With the plasmid DNA of preparation in the BamHI/EcoRI double digestion 1.5, the plasmid that occurs the DNA band of about 2.4kb, 0.7kb behind the electrophoresis is reorganization delivery plasmid pGEM-3Z (P1-2A).Cut the plasmid DNA for preparing in 1.5 with the EcoRI/BglII enzyme, the plasmid that occurs the DNA band of 0.7kb behind the electrophoresis is reorganization delivery plasmid pGEM-3Z (3C).Recombinant plasmid pGEM-3Z (P1-2A), pGEM-3Z (3C) are carried out two-way order-checking.P1-2A3C gene order and aminoacid sequence are seen SEQ ID NO:1 and SEQ ID NO:2 respectively.
2, the structure of transfer vector pVL1393 (P1-2A3C)
With plasmid pGEM-3Z (P1-2A) prepared in 1.6 with BamHI and EcoRI double digestion, with the method purifying P1-2A gene fragment in 1.2, with transformed into escherichia coli TG1 after baculovirus transfer vector pVL1393 through BamHI and EcoRI double digestion is connected, screening obtains recombinant transfer vector pVL1393 (P1-2A).With the plasmid pGEM-3Z (3C) of preparation in 1.6 with EcoRI and BglII double digestion, method purifying 3C gene fragment by 1.2, with transformed into escherichia coli TG1 after baculovirus transferring plasmid pVL1393 (P1-2A) through EcoRI and BglII double digestion is connected, screening obtains recombinant transfer vector pVL1393 (P1-2A3C).
3, the preparation of the breeding of silkworm nuclear-polyhedrosis virus parent plant Bm-NPV-ZJ8 and viral DNA
Press the description of product preparation 1 * TC-100 of GIBCO company substratum, with 2 N NaOH pH is transferred to 6.22, the substratum after the filtration sterilization is added 10% foetal calf serum, cultivates bombyx mori cell BmN down for 27 ℃.Infect the about 50ml of cell of logarithmic phase with silkworm nuclear-polyhedrosis virus parent plant Bm-NPV-ZJ8, infection multiplicity is 1, collect virus infection liquid after 3～4 days, centrifugal (5000rpm * 10min), remove precipitation, supernatant is used centrifugal 1 hour of 25000rpm, removes supernatant, (contains Tris 12.1g among the 1000ml with 1ml viral DNA extract, EDTA 33.6g, KCl 14.1g, pH7.5) suspension virus particle precipitation is transferred in the 1.5ml centrifuge tube, adding Proteinase K to final concentration is 50 μ g/ml, 50 ℃ of insulations 2 hours, add again 35% Sarkorsel to final concentration be 1%, continue at 50 ℃ of insulations 2 hours, use isopyknic phenol respectively, phenol: chloroform (1: 1) chloroform extracting successively, in upper water phase transition to a new pipe, add 3 M NaCl of 1/10 volume, add the dehydrated alcohol of 2 times of volumes again,-20 ℃ of placements precipitated viral DNA more than 2 hours, centrifugal 10 minutes of 5000rpm, precipitation is washed once lyophilize with 75% ethanol.Be dissolved among the 100 μ l TE Buffer, it is standby to put 4 ℃ of preservations.
4, structure and the acquisition of recombinant silkworm baculovirus rBmNPV (P1-2A3C)
4.1 the structure of recombinant baculovirus rBmNPV (P1-2A3C)
Inoculate about 1 * 10 6Cell is in 15cm 2In the culturing bottle, behind the cell attachment, remove and contain foetal calf serum (FBS) substratum, give a baby a bath on the third day after its birth time with the substratum that does not contain FBS, adding 1.5ml does not have the FBS substratum.In a sterile tube, add 1 μ g silkworm baculovirus parent plant Bm-NPV-ZJ8 DNA successively, 2 μ g recombinant transfer plasmid pVL1393 (P1-2A3C) DNA and 5 μ l liposomes, supply volume to 60 μ l with aseptic double-distilled water, mixing gently, after leaving standstill 15 minutes, dropwise join and carry out cotransfection in the culturing bottle.Cultivate after 4 hours for 27 ℃ and add 1.5ml serum free medium and 300 μ l FBS.27 ℃ of constant temperature culture 4～5 days are collected the screening that supernatant liquor is used for recombinant virus.
4.2 screening and the purifying of recombinant silkworm baculovirus rBmNPV (P1-2A3C)
Inoculate an amount of cell (about 70～80%) in the little plate of 35mm, behind the cell attachment, inhale and remove substratum, the cotransfection supernatant is carried out the different concns dilution, get 1ml corotation dye liquor and be added in the attached cell, be evenly distributed.27 ℃ were infected after 1 hour, suction removes to infect liquid, 2% low melting point sepharose is melted in 60 ℃ of water-baths, being chilled to 40 ℃ of 2 * TC-100 substratum (containing 20%FBS) with 40 ℃ of preheatings mixes, every plate adds 4ml glue, wait to solidify the back and seal, be inverted for 27 ℃ and cultivated microscopic examination 3～5 days with Parafilm.To not contain polyhedrosis plaque and pick out, and repeat above step, (its microbial preservation number is: CGMCC NO.1979) to obtain pure recombinant silkworm baculovirus rBmNPV (P1-2A3C) through 2～3 purifying of taking turns.
4.3 the amplification of recombinant virus rBmNPV (P1-2A3C) in bombyx mori cell
With the BmN cell of recombinant silkworm baculovirus rBmNPV (P1-2A3C) infection normal growth, cultivate and collect supernatant liquor after 3 days, promptly contain a large amount of recombinant virus rBmNPV (P1-2A3C) in the supernatant liquor.
4.4 the evaluation of recombinant virus
Utilize PCR method to analyze exogenous origin gene integrator.The extracting method of free virus genomic dna is as follows: get viral supernatant 150 μ l, add mixing behind the NaOH of 150 μ l (0.5mol/L), the ammonium acetate that adds 20 μ l (8mol/L) again, behind the mixing with isopyknic phenol and chloroform respectively extracting once, behind the alcohol precipitation with the TE dissolving DNA of 20 μ l.Oligonucleolide primers is:
5’-GAGGATCCACGATGAAAGCGATCTTAATCCCAT-3’
5’-TTACACCATCTGCTTTCCAGGTGCAAT-3’
Get above-mentioned virus genom DNA 1 μ l and carry out pcr amplification, reaction conditions is: 94 ℃ of sex change 5min, 94 ℃ of 1min, 58 ℃ of 1min, 72 ℃ of 1min, and 30 circulations, last 72 ℃ are extended 5min.Get 15 μ l reaction product electrophoretic analysiss, the result proves and has obtained recombinant virus.
5, the expression of P1-2A3C gene in silkworm
The used silkworm high expression level kind of this experiment is JY1 (being preserved by this laboratory).JY1 kind silkworm rearing is pressed the ordinary method of " Chinese sericulture " (Shanghai science tech publishing house, 1991) of Lv Hongsheng chief editor and is carried out.48h selects the identical silkworm of mean body weight behind the first feeding, and every silkworm inoculates about 1.0 * 10 5RBmNP (P1-2A3C) collects morbidity silkworm hemolymph after 4-5 days ,-20 ℃ frozen to carry out the detection of double-antibody sandwich elisa method.96 hole enzyme plates are with the anti-foot and mouth disease positive serum of rabbit bag quilt, and 4 ℃ are spent the night.After skim-milk seals, the silkworm blood that the positive antigen of foot and mouth disease, the silkworm blood that infects rBmNPV (P1-2A3C) silkworm results, the silkworm of infection Bm-NPV-ZJ8 are gathered in the crops is done 2 times of gradient dilutions.Behind 37 ℃ of effect 1h, washing adds the anti-FMDV positive serum of cavy, 37 ℃ of effect 1h.The washing back adds the anti-cavy IgG of HRP-rabbit, 37 ℃ of effect 1h.Add substrate OPD-H 2O 2, in 37 ℃ of effect 15min, finish reaction with stop buffer, measure the OD value in λ 492nm place.The hemolymph OD value that the result gathers in the crops from the silkworm that infects rBmNPV (P1-2A3C) as shown in Figure 1 descends gradually with the increase of doubling dilution degree, and its Changing Pattern is identical with positive control conventional cell seedling diseases poison antigen Changing Pattern.From OD pH-value determination pH experimental result, its expression amount reaches more than 100 times of antigenic expression of positive control conventional cell seedling diseases poison, and does not detect antigen presentation in the silkworm hemolymph that contrast Bm-NPV-ZJ8 infects.
6, the P1-2A3C gene is expressed in the silkworm pupa body
The used silkworm pupa of this experiment is JY1 (being preserved by this laboratory) for the high expression level kind.JY1 kind silkworm rearing is pressed the ordinary method of " Chinese sericulture " (Shanghai science tech publishing house, 1991) of Lv Hongsheng chief editor and is carried out.Selected 15 identical silkworm chrysalises of mean body weight after cocooing seven days, 48h selects the identical silkworm of mean body weight behind the first feeding, and every silkworm chrysalis inoculates about 1.0 * 10 5RBmNPV (P1-2A3C) collects morbidity silkworm chrysalis hemolymph after 4-5 days ,-20 ℃ frozen to carry out the detection of double-antibody sandwich elisa method.The hemolymph OD value that the result gathers in the crops from the silkworm pupa that infects rBmNPV (P1-2A3C) as shown in Figure 2 descends gradually with the increase of doubling dilution degree, and its Changing Pattern is identical with the antigenic OD value Changing Pattern of positive control conventional cell seedling diseases poison.From OD pH-value determination pH experimental result, its expression amount reaches more than 100 times of antigenic expression of positive control conventional cell seedling diseases poison, and does not detect antigen presentation in the silkworm hemolymph that contrast Bm-NPV-ZJ8 infects.
7, the purifying of foot-and-mouth disease antigen
Take by weighing the Sephadex xerogel, be loaded on after the swelling treatment in the glass column, (5mmol/LTris solution, 0.1mol/L NaCl pH8.0) are washed till baseline stability with elutriant.Respectively with the silkworm hemolymph gathered in the crops in above-mentioned 5 and 6 through ultrasonic disruption, the centrifugal cell debris that goes.Get sample on the above-mentioned sample, get an amount of elutriant, flow velocity 0.3ml/min collects the albumen elutriant with the 5min/ pipe, is collected into first peak and descends, and collected albumen elutriant is merged, and gets the foot-and-mouth disease antigen of purifying.
8, animal immune experiment and virus attack protection experiment
The purifying antigen of collecting is mixed with equal-volume oil adjuvant.Get 3ml/ incidence muscle immune cattle.5 oxen of experimental group immunity are set up the control group of 2 oxen.Before vaccinate, adopt of the detection of the standard method liquid phase blocking-up ELISA of OIE method to candidate Niu Jinhang antibody horizontal, select it and tire and be lower than 1/8 ox and experimentize at the isolation Animal House of Lanzhou veterinary institute
Collect the bovine serum of immunity back 21 d, employing liquid phase blocking-up ELISA method detects the anti-FMDV antibody horizontal in the bovine serum, and all laboratory animal have all been obtained higher antibody horizontal.Adopt 10,000BID 50Homology virus inoculation cow tongue, day-night observation 10d detects lip and four hoof incidences, 5 animals of experimental group have obtained whole protections, and control animals is all fallen ill.
The preparation of embodiment 2 foot-and-mouth disease antigens, purifying and animal immune experiment and virus attack protection experiment
1, the clone of foot and mouth disease virus total length ORF and sequential analysis
The design primer, the method by RT-PCR amplifies foot and mouth disease virus total length ORF (SEQ ID NO:3).
The amplimer of designed amplification total length ORF is,
ORF：5’-GCG ACTAGTACCATGGAATTCACACTTCACAACGGTGAG-3’，
Spe I restriction enzyme site
ORF：5’-ATA GCGGCCGCAGGGATTATGCGTCACCGCACAC-3’。
Not I restriction enzyme site
From the foot and mouth disease virus cell culture fluid, extract total RNA.With the total RNA Oligo (dT) that extracts 18Primer is under the effect of AMV ThermoScript II, and 42 ℃ of reverse transcriptions prepare cDNA.CDNA with acquisition is a template, carries out pcr amplification with Auele Specific Primer.
The PCR reaction system is as follows:
Table 2 PCR reaction conditions
Admixture Volume
Sterilization ddH 2O 33μl
10×PCR Buffer 5μl
2.5mM dNTP 4μl
25Mm MgCl 2 3μl
5 ' primers 1μl
3 ' primers 1μl
Admixture Volume
Template DNA 2μl
The Taq enzyme 1μl
Total 50μl
PCR reaction process: 94 ℃ of sex change 10 minutes; 94 ℃ 1 minute, 58 ℃ 1 minute, 72 ℃ 8 minutes, totally 30 circulations.Last extension 10 minutes.
2, the structure of transfer vector pVL1393 (ORF)
The ORF that middle amplification is obtained is by 1.2 method purifying among the embodiment 1, behind SpeI and NotI double digestion, with transformed into escherichia coli TG1 after baculovirus transfer vector pVL1393 through XbaI and NotI double digestion is connected, screening obtains recombinant transfer vector pVL1393 (ORF).Recombinant plasmid pVL1393 (ORF) is carried out two-way order-checking.ORF gene order and aminoacid sequence are seen SEQ ID NO:3 and SEQ ID NO:4 respectively.
3, structure and the acquisition of recombinant silkworm baculovirus rBmNPV (ORF)
3.1, the structure of recombinant baculovirus rBmNPV (ORF)
Inoculate about 1 * 10 6Cell is in 15cm 2In the culturing bottle, behind the cell attachment, remove and contain foetal calf serum (FBS) substratum, give a baby a bath on the third day after its birth time with the substratum that does not contain FBS, adding 1.5ml does not have the FBS substratum.In a sterile tube, add 1 μ g silkworm baculovirus parent plant Bm-NPV-ZJ8 DNA successively, 2 μ g recombinant transfer plasmid pVL1393 (ORF) DNA and 5 μ l liposomes, supply volume to 60 μ l with aseptic double-distilled water, mixing gently, after leaving standstill 15 minutes, dropwise join and carry out cotransfection in the culturing bottle.Cultivate after 4 hours for 27 ℃ and add 1.5 ml serum free mediums and 300 μ l FBS.27 ℃ of constant temperature culture 4～5 days are collected the screening that supernatant liquor is used for recombinant virus.
3.2 screening and the purifying of recombinant silkworm baculovirus rBmNPV (ORF)
Inoculate an amount of cell (about 70～80%) in the little plate of 35mm, behind the cell attachment, inhale and remove substratum, the cotransfection supernatant is carried out the different concns dilution, get 1ml corotation dye liquor and be added in the attached cell, be evenly distributed.27 ℃ were infected after 1 hour, suction removes to infect liquid, 2% low melting point sepharose is melted in 60 ℃ of water-baths, being chilled to 40 ℃ of 2 * TC-100 substratum (containing 20%FBS) with 40 ℃ of preheatings mixes, every plate adds 4ml glue, wait to solidify the back and seal, be inverted for 27 ℃ and cultivated microscopic examination 3～5 days with Parafilm.To not contain polyhedrosis plaque and pick out, and repeat above step, (its microbial preservation number is: CGMCC NO.1980) to obtain pure recombinant silkworm baculovirus rBmNPV (ORF) through 2～3 purifying of taking turns.
3.3 the amplification of recombinant virus rBmNPV (ORF) in bombyx mori cell
With the BmN cell of recombinant silkworm baculovirus rBmNPV (ORF) infection normal growth, cultivate and collect supernatant liquor after 3 days, promptly contain a large amount of recombinant virus rBmNPV (ORF) in the supernatant liquor.
3.4 the evaluation of recombinant virus
Utilize PCR method to analyze exogenous origin gene integrator.The extracting method of free virus genomic dna is as follows: get viral supernatant 150 μ l, add mixing behind the NaOH of 150 μ l (0.5mol/L), the ammonium acetate that adds 20 μ l (8mol/L) again, behind the mixing with isopyknic phenol and chloroform respectively extracting once, behind the alcohol precipitation with the TE dissolving DNA of 20 μ l.Oligonucleolide primers is:
5’-GAGGATCCACGATGAAAGCGATCTTAATCCCAT-3’
5’-TTACACCATCTGCTTTCCAGGTGCAAT-3’
Get above-mentioned virus genom DNA 1 μ l and carry out pcr amplification, reaction conditions is: 94 ℃ of sex change 5min, 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min, and 30 circulations, last 72 ℃ are extended 5min.Get 15 μ l reaction product electrophoretic analysiss, the result proves and has obtained recombinant virus.
4, the expression of ORF in silkworm
The used silkworm high expression level kind of this experiment is JY1 (being preserved by this laboratory).JY1 kind silkworm rearing is pressed the ordinary method of " Chinese sericulture " (Shanghai science tech publishing house, 1991) of Lv Hongsheng chief editor and is carried out.48h selects the identical silkworm of mean body weight behind the first feeding, and every silkworm inoculates about 1.0 * 10 5RBmNPV (ORF) collects morbidity silkworm hemolymph after 4-5 days ,-20 ℃ frozen to carry out the detection of double-antibody sandwich elisa method.96 hole enzyme plates are with the anti-FMDV positive serum of rabbit bag quilt, and 4 ℃ are spent the night.After the skim-milk sealing, the silkworm blood that FMDV antigen, the silkworm blood that infects rBmNPV (ORF) silkworm results, the silkworm of infection Bm-NPV-ZJ8 are gathered in the crops is done 2 times of gradient dilutions.Behind 37 ℃ of effect 1h, washing adds the anti-FMDV positive serum of cavy, 37 ℃ of effect 1h.The washing back adds the anti-cavy IgG of HRP-rabbit, 37 ℃ of effect 1h.Add substrate OPD-H 2O 2, in 37 ℃ of effect 15min, finish reaction with stop buffer, measure the OD value in λ 492nm place.The hemolymph OD value that the result gathers in the crops from the silkworm that infects rBmNPV (ORF) as shown in Figure 3 descends gradually with the increase of doubling dilution degree, and its Changing Pattern is identical with the antigenic OD value Changing Pattern of positive control conventional cell seedling diseases poison.From OD pH-value determination pH experimental result, its expression amount reaches more than 10 times of antigenic expression of positive control conventional cell seedling diseases poison, and does not detect antigen presentation in the silkworm hemolymph that contrast Bm-NPV-ZJ8 infects.
5, ORF expresses in the silkworm pupa body
The used silkworm pupa of this experiment is JY1 (being preserved by this laboratory) for the high expression level kind.JY1 kind silkworm rearing is pressed the ordinary method of " Chinese sericulture " (Shanghai science tech publishing house, 1991) of Lv Hongsheng chief editor and is carried out.Selected 15 identical silkworm chrysalises of mean body weight after cocooing seven days, 48h selects the identical silkworm chrysalis of mean body weight behind the first feeding, and every silkworm chrysalis inoculates about 1.0 * 10 5Pfu rBmNPV (ORF) collects morbidity silkworm chrysalis hemolymph after 4-5 days ,-20 ℃ frozen to carry out the detection of double-antibody sandwich elisa method.The hemolymph OD value that the result gathers in the crops from the silkworm chrysalis of sense infection rBmNPV (ORF) as shown in Figure 4 descends gradually with the increase of doubling dilution degree, and its Changing Pattern is identical with positive control conventional cell seedling diseases poison antigen Changing Pattern.From OD pH-value determination pH experimental result, its expression amount reaches more than 10 times of antigenic expression of positive control conventional cell seedling diseases poison, and does not detect antigen presentation in the silkworm hemolymph that contrast Bm-NPV-ZJ8 infects.
6, antigen purification
Take by weighing the Sephadex xerogel, be loaded on after the swelling treatment in the glass column, (5mmol/LTris solution, 0.1mol/L NaCl pH8.0) are washed till baseline stability with elutriant.Respectively with the 4 and 5 silkworm hemolymphs of being gathered in the crops through ultrasonic disruption, the centrifugal cell debris that goes.Get sample on the above-mentioned sample, get an amount of elutriant, flow velocity 0.3ml/min collects the albumen elutriant with the 5min/ pipe, is collected into first peak and descends, and merges the albumen elutriant, gets purifying antigen.
7, animal immune experiment and virus attack protection experiment
6 collected purifying antigens are mixed with equal-volume oil adjuvant.Get 3ml/ incidence muscle immune cattle.5 oxen of experimental group immunity are set up the control group of 2 oxen.Before vaccinate, adopt of the detection of the standard method liquid phase blocking-up ELISA of OIE method to candidate Niu Jinhang antibody horizontal, select it and tire and be lower than 1/8 ox and experimentize at the isolation Animal House of Lanzhou veterinary institute.
Collect the bovine serum of immunity back 21d, employing liquid phase blocking-up ELISA method detects the anti-FMDV antibody horizontal in the bovine serum, and all laboratory animal have all been obtained higher antibody horizontal.Adopt 10,000BID 50Homology virus inoculation cow tongue, day-night observation 10d detects lip and four hoof incidences, 5 animals of experimental group have obtained 4 head protections, and control animals is all fallen ill.
The preparation of embodiment 3 foot-and-mouth disease antigens, purifying and animal immune experiment and virus attack protection experiment
1, the clone of FMDV VP1 gene and sequential analysis
The design primer, the method by RT-PCR amplifies foot and mouth disease virus VP1 gene.
The amplimer of designed amplification VP1 gene is,
VP1 upstream: 5 '-ATA GGATCCACCATGGCCACCACTACCGGCGAGTCAG-3 ',
BamH I restriction enzyme site
VP1 downstream: 5 '-CGC GAATTCTTACACCATCTGCTTTCCAGGTGCAAT-3 '.
EcoR I restriction enzyme site
From the foot and mouth disease virus cell culture fluid, extract total RNA.With the total RNA Oligo (dT) that extracts 18Primer is under the effect of AMV ThermoScript II, and 42 ℃ of reverse transcriptions prepare cDNA.CDNA with acquisition is a template, carries out pcr amplification with Auele Specific Primer.
The PCR reaction system is as follows:
Table 3PCR reaction conditions
Admixture Volume
Sterilization ddH 2O 33μl
10×PCR Buffer 5μl
2.5 Mm dNTP 4μl
25 MmMgCl 2 3μl
5 ' primers 1μl
3 ' primers 1μl
Template DNA 2μl
The Taq enzyme 1μl
Total 50μl
PCR reaction process: 94 ℃ of sex change 10 minutes; 94 ℃ 1 minute, 58 ℃ 1 minute, 72 ℃ 1 minute, totally 30 circulations.Last extension 10 minutes.
2, the structure of transfer vector pVL1393 (VP1)
Press among the embodiment 1 1.2 the method purifying 1 VP1 gene fragment that obtains, behind BamHI and EcoRI double digestion, with transformed into escherichia coli TG1 after the baculovirus transfer vector pVL1393 of double digestion is connected equally, screening obtains reorganization and shifts a year pVL1393 (VP1).Recombinant plasmid pVL1393 (VP1) is carried out two-way order-checking.VP1 gene order and aminoacid sequence are seen SEQ ID NO:5 and SEQ ID NO:6 respectively.
3, structure and the acquisition of recombinant silkworm baculovirus rBmNPV (VP1)
3.1 the structure of recombinant baculovirus rBmNPV (VP1)
Inoculate about 1 * 10 6Cell is in 15cm 2In the culturing bottle, behind the cell attachment, remove and contain foetal calf serum (FBS) substratum, give a baby a bath on the third day after its birth time with the substratum that does not contain FBS, adding 1.5ml does not have the FBS substratum.In a sterile tube, add 1 μ g silkworm baculovirus parent plant Bm-NPV-ZJ8 DNA successively, 2 μ g recombinant transfer plasmid pVL1393 (VP1) DNA and 5 μ l liposomes, supply volume to 60 μ l with aseptic double-distilled water, mixing gently, after leaving standstill 15 minutes, dropwise join and carry out cotransfection in the culturing bottle.Cultivate after 4 hours for 27 ℃ and add 1.5ml serum free medium and 300 μ l FBS.27 ℃ of constant temperature culture 4～5 days are collected the screening that supernatant liquor is used for recombinant virus.
3.2 screening and the purifying of recombinant silkworm baculovirus rBmNPV (VP1)
Inoculate an amount of cell (about 70～80%) in the little plate of 35mm, behind the cell attachment, inhale and remove substratum, the cotransfection supernatant is carried out the different concns dilution, get 1ml corotation dye liquor and be added in the attached cell, be evenly distributed.27 ℃ were infected after 1 hour, suction removes to infect liquid, 2% low melting point sepharose is melted in 60 ℃ of water-baths, being chilled to 40 ℃ of 2 * TC-100 substratum (containing 20%FBS) with 40 ℃ of preheatings mixes, every plate adds 4ml glue, wait to solidify the back and seal, be inverted for 27 ℃ and cultivated microscopic examination 3～5 days with Parafilm.To not contain polyhedrosis plaque and pick out, and repeat above step, (its microbial preservation number is: CGMCC NO.1975) to obtain pure recombinant silkworm baculovirus rBmNPV (VP1) through 2～3 purifying of taking turns.
3.3 the amplification of recombinant virus rBmNPV (VP1) in bombyx mori cell
With the BmN cell of recombinant silkworm baculovirus rBmNPV (VP1) infection normal growth, cultivate and collect supernatant liquor after 3 days, promptly contain a large amount of recombinant virus rBmNPV (VP1) in the supernatant liquor.
3.4 the evaluation of recombinant virus
Utilize PCR method to analyze exogenous origin gene integrator.The extracting method of free virus genomic dna is as follows: get viral supernatant 150 μ l, add mixing behind the NaOH of 150 μ l (0.5mol/L), the ammonium acetate that adds 20 μ l (8mol/L) again, behind the mixing with isopyknic phenol and chloroform respectively extracting once, behind the alcohol precipitation with the TE dissolving DNA of 20 μ l.Oligonucleolide primers is:
5’-GAGGATCCACGATGAAAGCGATCTTAATCCCAT-3’
5’-TTACACCATCTGCTTTCCAGGTGCAAT-3’
Get above-mentioned virus genom DNA 1 μ l and carry out pcr amplification, reaction conditions is: 94 ℃ of sex change 5min, 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min, and 30 circulations, last 72 ℃ are extended 5min.Get 15 μ l reaction product electrophoretic analysiss, the result proves and has obtained recombinant virus.
4, the expression of VP1 in silkworm
The used silkworm high expression level kind of this experiment is JY1 (being preserved by this laboratory).JY1 kind silkworm rearing is pressed the ordinary method of " Chinese sericulture " (Shanghai science tech publishing house, 1991) of Lv Hongsheng chief editor and is carried out.48h selects the identical silkworm of mean body weight behind the first feeding, and every silkworm inoculates about 1.0 * 10 5RBmNPV (VP1) collects morbidity silkworm hemolymph after 4-5 days ,-20 ℃ frozen to carry out the detection of double-antibody sandwich elisa method.The hemolymph OD value that the result gathers in the crops from the silkworm that infects rBmNPV (VP1) as shown in Figure 5 descends gradually with the increase of doubling dilution degree, and its Changing Pattern is positive identical with contrast conventional cell seedling diseases poison antigen Changing Pattern.From OD pH-value determination pH experimental result, its expression amount reaches more than 100 times of antigenic expression of positive control conventional cell seedling diseases poison, and does not detect antigen presentation in the silkworm hemolymph that contrast Bm-NPV-ZJ8 infects.
5, the VP1 gene is expressed in the silkworm pupa body
The used silkworm pupa of this experiment is JY1 (being preserved by this laboratory) for the high expression level kind.JY1 kind silkworm rearing is pressed the ordinary method of " Chinese sericulture " (Shanghai science tech publishing house, 1991) of Lv Hongsheng chief editor and is carried out.Selected 15 identical silkworm chrysalises of mean body weight after cocooing seven days, 48h selects the identical silkworm chrysalis of mean body weight behind the first feeding, and every silkworm chrysalis inoculates about 1.0 * 10 5RBmNPV (VP1) collects morbidity silkworm hemolymph after 4-5 days ,-20 ℃ frozen to carry out the detection of double-antibody sandwich elisa method.The hemolymph OD value that the result gathers in the crops from the silkworm that infects rBmNPV (ORF) as shown in Figure 6 descends gradually with the increase of doubling dilution degree, and its Changing Pattern is identical with positive control conventional cell seedling diseases poison antigen Changing Pattern.From OD pH-value determination pH experimental result, its expression amount reaches more than 100 times of antigenic expression of positive control conventional cell seedling diseases poison, and does not detect antigen presentation in the silkworm hemolymph that contrast Bm-NPV-ZJ8 infects.
6, antigen purification
Take by weighing the Sephadex xerogel, be loaded on after the swelling treatment in the glass column, be washed till baseline stability with elutriant (pH 8.0 for 5mmol/LTris solution, 0.1mol/LNaCl).Respectively with the 4 and 5 silkworm hemolymphs of being gathered in the crops through ultrasonic disruption, the centrifugal cell debris that goes.Get sample on the above-mentioned sample, get an amount of elutriant, flow velocity 0.3ml/min collects the albumen elutriant with the 5min/ pipe, is collected into first peak and descends, and merges the albumen elutriant, gets purifying antigen.
7, animal immune experiment and virus attack protection experiment
With the 4 or 5 silkworm hemolymphs of collecting through ultrasonic disruption, abandon cell debris after, mix with equal-volume oil adjuvant.Get 3ml/ incidence muscle immune cattle.5 oxen of experimental group immunity are set up the control group of 2 oxen.Before vaccinate, adopt of the detection of the standard method liquid phase blocking-up ELISA of OIE method to candidate Niu Jinhang antibody horizontal, select it and tire and be lower than 1/8 ox and experimentize at the isolation Animal House of Lanzhou veterinary institute
Collect the bovine serum of immunity back 21d, employing liquid phase blocking-up ELISA method detects the anti-foot-and-mouth disease antibody level in the bovine serum, and all laboratory animal have all been obtained higher antibody horizontal.Adopt 10,000BID 50Homology virus inoculation cow tongue, day-night observation 10d detects lip and four hoof incidences, 5 animals of experimental group have 4 to be protected, and control animals is all fallen ill.
Sequence table
＜110〉Biological Technology institute, Chinese Academy of Agricultural Sciences of Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
＜120〉a kind of preparation method of foot-and-mouth disease antigen
<130>P0278
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<170>PatentIn version 3.1
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<213>Foot-and-mouth disease virus
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gga gcc ggg caa tcc agc ccg gcg act ggg tca cag aac cag tca ggc 48
Gly Ala Gly Gln Ser Ser Pro Ala Thr Gly Ser Gln Asn Gln Ser Gly
1 5 10 15
aac act gga agc atc atc aac aac tac tac atg cag caa tac cag aac 96
Asn Thr Gly Ser Ile Ile Asn Asn Tyr Tyr Met Gln Gln Tyr Gln Asn
20 25 30
tcc atg gac aca caa ctt gga gac aac gct att agc gga ggc tcc aac 144
Ser Met Asp Thr Gln Leu Gly Asp Asn Ala Ile Ser Gly Gly Ser Asn
35 40 45
gaa ggt tcc act gac acc acc tcc aca cac aca aac act acc caa aac 192
Glu Gly Ser Thr Asp Thr Thr Ser Thr His Thr Asn Thr Thr Gln Asn
50 55 60
aac gat tgg ttc tcg cgc ctg gct agt tcc gcc ttc agc gga ctg ttc 240
Asn Asp Trp Phe Ser Arg Leu Ala Ser Ser Ala Phe Ser Gly Leu Phe
65 70 75 80
ggc gcg ctt ctg gct gac aag aaa acg gaa gaa aca act ttg ctt gaa 288
Gly Ala Leu Leu Ala Asp Lys Lys Thr Glu Glu Thr Thr Leu Leu Glu
85 90 95
gac cgc atc ctt acc acc agg aac ggc cac acg acg tcg acg acc cag 336
Asp Arg Ile Leu Thr Thr Arg Asn Gly His Thr Thr Ser Thr Thr Gln
100 105 110
tcg agc gtt ggc gtg aca tac ggt tac gct gtg gct gag gac gcg gta 384
Ser Ser Val Gly Val Thr Tyr Gly Tyr Ala Val Ala Glu Asp Ala Val
115 120 125
tca gga cct aac acc tca ggt ctc gag acc cgt gtt caa caa gcg gag 432
Ser Gly Pro Asn Thr Ser Gly Leu Glu Thr Arg Val Gln Gln Ala Glu
130 135 140
cgg ttc ttc aaa aag cac ttg ttt gat tgg aca ccg aat ttg gct ttt 480
Arg Phe Phe Lys Lys His Leu Phe Asp Trp Thr Pro Asn Leu Ala Phe
145 150 155 160
gga tac tgt cac tac ctg gaa ctc ccc act gag cac aaa ggt gtg tat 528
Gly Tyr Cys His Tyr Leu Glu Leu Pro Thr Glu His Lys Gly Val Tyr
165 170 175
ggc agt ctc atg gac tcg tac gcc tac atg aga aac ggg tgg gac ata 576
Gly Ser Leu Met Asp Ser Tyr Ala Tyr Met Arg Asn Gly Trp Asp Ile
180 185 190
gag gtg act gct gtt gga aac cag ttc aac ggc ggt tgt ctc ctt gtt 624
Glu Val Thr Ala Val Gly Asn Gln Phe Asn Gly Gly Cys Leu Leu Val
195 200 205
gca ctt gtg cca gag ctg aag agc ctt gac act cgg cag aaa tac caa 672
Ala Leu Val Pro Glu Leu Lys Ser Leu Asp Thr Arg Gln Lys Tyr Gln
210 215 220
ctg acc ctt ttc ccc cat cag ttc atc aac cca cgc acc aac atg acg 720
Leu Thr Leu Phe Pro His Gln Phe Ile Asn Pro Arg Thr Asn Met Thr
225 230 235 240
gcc cac atc aac gtg ccc ttt ata ggt gtt aac agg tat gac cag tac 768
Ala His Ile Asn Val Pro Phe Ile Gly Val Asn Arg Tyr Asp Gln Tyr
245 250 255
atg ctc cac aaa ccg tgg acg ctt gtt gtg atg gtg gtg gcc cca ctc 816
Met Leu His Lys Pro Trp Thr Leu Val Val Met Val Val Ala Pro Leu
260 265 270
act gtc aag act ggt ggc tct gaa cag atc aag gtc tac atg aat gca 864
Thr Val Lys Thr Gly Gly Ser Glu Gln Ile Lys Val Tyr Met Asn Ala
275 280 285
gca ccg acc tac gta cac gtg gca ggg gag ctc ccc tcg aaa gag ggg 912
Ala Pro Thr Tyr Val His Val Ala Gly Glu Leu Pro Ser Lys Glu Gly
290 295 300
ata gtt ccc gtt gcg tgt gcg gac ggt tac ggt aac atg gtg acc acg 960
Ile Val Pro Val Ala Cys Ala Asp Gly Tyr Gly Asn Met Val Thr Thr
305 310 315 320
gac ccg aaa act gcc gac cca gtg tac ggg aaa gtg ttc aac ccc ccc 1008
Asp Pro Lys Thr Ala Asp Pro Val Tyr Gly Lys Val Phe Asn Pro Pro
325 330 335
cgg acg aat ctc ccc ggg cgc ttc aca aac ttc ctt gat gtc gcg gag 1056
Arg Thr Asn Leu Pro Gly Arg Phe Thr Asn Phe Leu Asp Val Ala Glu
340 345 350
gcg tgt cca acc ttc ctc cgc ttc gga gaa gta cca ttt gtg aag acg 1104
Ala Cys Pro Thr Phe Leu Arg Phe Gly Glu Val Pro Phe Val Lys Thr
355 360 365
gtg aac tct ggt gac cgt ttg cta gcc aag ttt gat gtc tcg ctc gct 1152
Val Asn Ser Gly Asp Arg Leu Leu Ala Lys Phe Asp Val Ser Leu Ala
370 375 380
gcg ggc cat atg tcc aac acc tac ttg gct ggt ctg gcg cag tac tac 1200
Ala Gly His Met Ser Asn Thr Tyr Leu Ala Gly Leu Ala Gln Tyr Tyr
385 390 395 400
aca cag tac agt ggt acc atg aat gtt cac ttc atg ttc acc ggg ccc 1248
Thr Gln Tyr Ser Gly Thr Met Asn Val His Phe Met Phe Thr Gly Pro
405 410 415
acg gat gcc aag gcc cgg tac atg gtg gcc tac att cct ccc ggc atg 1296
Thr Asp Ala Lys Ala Arg Tyr Met Val Ala Tyr Ile Pro Pro Gly Met
420 425 430
aca ccg ccc aca gac cct gag cgc gcc gcc cac tgt atc cac tcc gag 1344
Thr Pro Pro Thr Asp Pro Glu Arg Ala Ala His Cys Ile His Ser Glu
435 440 445
tgg gac act ggt cta aac tcc aaa ttc acc ttt tcc ata ccc tac ctc 1392
Trp Asp Thr Gly Leu Asn Ser Lys Phe Thr Phe Ser Ile Pro Tyr Leu
450 455 460
tct gct gct gac tac gca tac acc gcc tct gac acg gcg gag acc aca 1440
Ser Ala Ala Asp Tyr Ala Tyr Thr Ala Ser Asp Thr Ala Glu Thr Thr
465 470 475 480
agt gtg caa gga tgg gtg tgc atc tac cag atc acc cac ggc aag gct 1488
Ser Val Gln Gly Trp Val Cys Ile Tyr Gln Ile Thr His Gly Lys Ala
485 490 495
gaa gga gac gca ctg gtc gtt tct gtc agc gcc ggc aaa gac ttt gag 1536
Glu Gly Asp Ala Leu Val Val Ser Val Ser Ala Gly Lys Asp Phe Glu
500 505 510
ttt cgc ctg ccc gtt gac gcg cgc cgg caa acc acc act acc ggc gag 1584
Phe Arg Leu Pro Val Asp Ala Arg Arg Gln Thr Thr Thr Thr Gly Glu
515 520 525
tca gca gac ccg gta aca acc acg gtc gag aac tac gga gga gaa act 1632
Ser Ala Asp Pro Val Thr Thr Thr Val Glu Asn Tyr Gly Gly Glu Thr
530 535 540
cag aca gcc agg cgg ctt cac act gat gtt gcc ttc gtt ctt gac agg 1680
Gln Thr Ala Arg Arg Leu His Thr Asp Val Ala Phe Val Leu Asp Arg
545 550 555 560
ttt gtg aaa ctc act gca ccc aag aac att cag acc ctt gac ctc atg 1728
Phe Val Lys Leu Thr Ala Pro Lys Asn Ile Gln Thr Leu Asp Leu Met
565 570 575
caa att ccc tca cac acg ctg gtt gga gca ctg ctg cgg tct gcg acg 1776
Gln Ile Pro Ser His Thr Leu Val Gly Ala Leu Leu Arg Ser Ala Thr
580 585 590
tac tac ttc tca gac ctg gag gtt gcg att gtc cac aca ggc ccg atc 1824
Tyr Tyr Phe Ser Asp Leu Glu Val Ala Ile Val His Thr Gly Pro Ile
595 600 605
acc tgg gtg ccc aac ggc tcg ccc aag gat gct cta gac aac cag acc 1872
Thr Trp Val Pro Asn Gly Ser Pro Lys Asp Ala Leu Asp Asn Gln Thr
610 615 620
aac cca act gcc tac cag aag caa cct gtc acc cgc ttg gcg ctc ccc 1920
Asn Pro Thr Ala Tyr Gln Lys Gln Pro Val Thr Arg Leu Ala Leu Pro
625 630 635 640
tac acc gcc ccc cat cgt gtg ctg gcg aca gtg tac aac ggg aag acg 1968
Tyr Thr Ala Pro His Arg Val Leu Ala Thr Val Tyr Asn Gly Lys Thr
645 650 655
acg tac ggg gaa aca acc gcg cgg cgt ggc gat acg gcg gcc ctt gca 2016
Thr Tyr Gly Glu Thr Thr Ala Arg Arg Gly Asp Thr Ala Ala Leu Ala
660 665 670
caa aga ctg agt ggg cgg ctg ccc acc tca ttt aac tac ggc gct gta 2064
Gln Arg Leu Ser Gly Arg Leu Pro Thr Ser Phe Asn Tyr Gly Ala Val
675 680 685
aag gct gaa acc atc act gag ctt ttg att cgc atg aaa cgt gcg gag 2112
Lys Ala Glu Thr Ile Thr Glu Leu Leu Ile Arg Met Lys Arg Ala Glu
690 695 700
aca tac tgc cct agg cct ttg cta gct ctt gac acc act cag gac cgc 2160
Thr Tyr Cys Pro Arg Pro Leu Leu Ala Leu Asp Thr Thr Gln Asp Arg
705 710 715 720
cgt aaa cag aag atc att gca cct gga aag cag atg gtg aac ttt gac 2208
Arg Lys Gln Lys Ile Ile Ala Pro Gly Lys Gln Met Val Asn Phe Asp
725 730 735
ctg ctc aag ttg gca gga gac gtc gag tcc aac cct ggg ccc ttc ttc 2256
Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro Phe Phe
740 745 750
ttc tcc gac gtc agg tcg aat ttt tcc aag ctg gtt gaa acc atc aac 2304
Phe Ser Asp Val Arg Ser Asn Phe Ser Lys Leu Val Glu Thr Ile Asn
755 760 765
cag atg cag gag gac atg tca gaa ttc aag aaa cct gtc gct ttg aaa 2352
Gln Met Gln Glu Asp Met Ser Glu Phe Lys Lys Pro Val Ala Leu Lys
770 775 780
gtg aaa gca aag aac ttg att gtc act gag agt ggt gct ccc ccg act 2400
Val Lys Ala Lys Asn Leu Ile Val Thr Glu Ser Gly Ala Pro Pro Thr
785 790 795 800
gac ttg caa aag atg gtc atg ggt aac acc aag cct gtt gag ctc atc 2448
Asp Leu Gln Lys Met Val Met Gly Asn Thr Lys Pro Val Glu Leu Ile
805 810 815
ctc gac ggg aag acg gtg gcc atc tgc cgc gcc acc gga gtg ttt ggt 2496
Leu Asp Gly Lys Thr Val Ala Ile Cys Arg Ala Thr Gly Val Phe Gly
820 825 830
act gcc tac ctt gtt cct cgt cat ctc ttc gca gag aag tat gac gag 2544
Thr Ala Tyr Leu Val Pro Arg His Leu Phe Ala Glu Lys Tyr Asp Glu
835 840 845
atc atg ctg gac ggc aga gcc atg aca gac agt gac tac aga gtg ttt 2592
Ile Met Leu Asp Gly Arg Ala Met Thr Asp Ser Asp Tyr Arg Val Phe
850 855 860
gag ttt gag att aaa gta aaa gga cag gac atg ctc tca gac gcc gcg 2640
Glu Phe Glu Ile Lys Val Lys Gly Gln Asp Met Leu Ser Asp Ala Ala
865 870 875 880
ctc atg gtg ctc cac cgt ggg aat cgc gtg cgg gac atc acg aag cac 2688
Leu Met Val Leu His Arg Gly Asn Arg Val Arg Asp Ile Thr Lys His
885 890 895
ttc cgt gat gtg gca aga atg cag aaa ggc acc ccc gtc gtc ggc gta 2736
Phe Arg Asp Val Ala Arg Met Gln Lys Gly Thr Pro Val Val Gly Val
900 905 910
atc aac aac gct gat gtt ggg aga ctg att ttc tct ggt gag gcc ctt 2784
Ile Asn Asn Ala Asp Val Gly Arg Leu Ile Phe Ser Gly Glu Ala Leu
915 920 925
acc tac aag gac att gta gtg tgc atg gac gga gac acc atg ccc ggc 2832
Thr Tyr Lys Asp Ile Val Val Cys Met Asp Gly Asp Thr Met Pro Gly
930 935 940
ctc ttc gcc tac aaa gct gcc acc aag gca ggt tac tgt gga gga gcc 2880
Leu Phe Ala Tyr Lys Ala Ala Thr Lys Ala Gly Tyr Cys Gly Gly Ala
945 950 955 960
gtt ctt gca aag gac gga gcc gag act ttc atc gtt ggc act cac tct 2928
Val Leu Ala Lys Asp Gly Ala Glu Thr Phe Ile Val Gly Thr His Ser
965 970 975
gca ggt ggc aat gga gtt gga tac tgc tca tgc gtt tcc agg tct atg 2976
Ala Gly Gly Asn Gly Val Gly Tyr Cys Ser Cys Val Ser Arg Ser Met
980 985 990
ctg ctc aaa atg aag gca cac atc gat ccc gaa cca cac cac gag tag 3024
Leu Leu Lys Met Lys Ala His Ile Asp Pro Glu Pro His His Glu
995 1000 1005
<210>2
<211>1007
<212>PRT
<213>Foot-and-mouth disease virus
<400>2
Gly Ala Gly Gln Ser Ser Pro Ala Thr Gly Ser Gln Asn Gln Ser Gly
1 5 10 15
Asn Thr Gly Ser Ile Ile Asn Asn Tyr Tyr Met Gln Gln Tyr Gln Asn
20 25 30
Ser Met Asp Thr Gln Leu Gly Asp Asn Ala Ile Ser Gly Gly Ser Asn
35 40 45
Glu Gly Ser Thr Asp Thr Thr Ser Thr His Thr Asn Thr Thr Gln Asn
50 55 60
Asn Asp Trp Phe Ser Arg Leu Ala Ser Ser Ala Phe Ser Gly Leu Phe
65 70 75 80
Gly Ala Leu Leu Ala Asp Lys Lys Thr Glu Glu Thr Thr Leu Leu Glu
85 90 95
Asp Arg Ile Leu Thr Thr Arg Asn Gly His Thr Thr Ser Thr Thr Gln
100 105 110
Ser Ser Val Gly Val Thr Tyr Gly Tyr Ala Val Ala Glu Asp Ala Val
115 120 125
Ser Gly Pro Asn Thr Ser Gly Leu Glu Thr Arg Val Gln Gln Ala G1u
130 135 140
Arg Phe Phe Lys Lys His Leu Phe Asp Trp Thr Pro Asn Leu Ala Phe
145 150 155 160
Gly Tyr Cys His Tyr Leu Glu Leu Pro Thr Glu His Lys Gly Val Tyr
165 170 175
Gly Ser Leu Met Asp Ser Tyr Ala Tyr Met Arg Asn Gly Trp Asp Ile
180 185 190
Glu Val Thr Ala Val Gly Asn Gln Phe Asn Gly Gly Cys Leu Leu Val
195 200 205
Ala Leu Val Pro Glu Leu Lys Ser Leu Asp Thr Arg Gln Lys Tyr Gln
210 215 220
Leu Thr Leu Phe Pro His Gln Phe Ile Asn Pro Arg Thr Asn Met Thr
225 230 235 240
Ala His Ile Asn Val Pro Phe Ile Gly Val Asn Arg Tyr Asp Gln Tyr
245 250 255
Met Leu His Lys Pro Trp Thr Leu Val Val Met Val Val Ala Pro Leu
260 265 270
Thr Val Lys Thr Gly Gly Ser Glu Gln Ile Lys Val Tyr Met Asn Ala
275 280 285
Ala Pro Thr Tyr Val His Val Ala Gly Glu Leu Pro Ser Lys Glu Gly
290 295 300
Ile Val Pro Val Ala Cys Ala Asp Gly Tyr Gly Asn Met Val Thr Thr
305 310 315 320
Asp Pro Lys Thr Ala Asp Pro Val Tyr Gly Lys Val Phe Asn Pro Pro
325 330 335
Arg Thr Asn Leu Pro Gly Arg Phe Thr Asn Phe Leu Asp Val Ala Glu
340 345 350
Ala Cys Pro Thr Phe Leu Arg Phe Gly Glu Val Pro Phe Val Lys Thr
355 360 365
Val Asn Ser Gly Asp Arg Leu Leu Ala Lys Phe Asp Val Ser Leu Ala
370 375 380
Ala Gly His Met Ser Asn Thr Tyr Leu Ala Gly Leu Ala Gln Tyr Tyr
385 390 395 400
Thr Gln Tyr Ser Gly Thr Met Asn Val His Phe Met Phe Thr Gly Pro
405 410 415
Thr Asp Ala Lys Ala Arg Tyr Met Val Ala Tyr Ile Pro Pro Gly Met
420 425 430
Thr Pro Pro Thr Asp Pro Glu Arg Ala Ala His Cys Ile His Ser Glu
435 440 445
Trp Asp Thr Gly Leu Asn Ser Lys Phe Thr Phe Ser Ile Pro Tyr Leu
450 455 460
Ser Ala Ala Asp Tyr Ala Tyr Thr Ala Ser Asp Thr Ala Glu Thr Thr
465 470 475 480
Ser Val Gln Gly Trp Val Cys Ile Tyr Gln Ile Thr His Gly Lys Ala
485 490 495
Glu Gly Asp Ala Leu Val Val Ser Val Ser Ala Gly Lys Asp Phe Glu
500 505 510
Phe Arg Leu Pro Val Asp Ala Arg Arg Gln Thr Thr Thr Thr Gly Glu
515 520 525
Ser Ala Asp Pro Val Thr Thr Thr Val Glu Asn Tyr Gly Gly Glu Thr
530 535 540
Gln Thr Ala Arg Arg Leu His Thr Asp Val Ala Phe Val Leu Asp Arg
545 550 555 560
Phe Val Lys Leu Thr Ala Pro Lys Asn Ile Gln Thr Leu Asp Leu Met
565 570 575
Gln Ile Pro Ser His Thr Leu Val Gly Ala Leu Leu Arg Ser Ala Thr
580 585 590
Tyr Tyr Phe Ser Asp Leu Glu Val Ala Ile Val His Thr Gly Pro Ile
595 600 605
Thr Trp Val Pro Asn Gly Ser Pro Lys Asp Ala Leu Asp Asn Gln Thr
610 615 620
Asn Pro Thr Ala Tyr Gln Lys Gln Pro Val Thr Arg Leu Ala Leu Pro
625 630 635 640
Tyr Thr Ala Pro His Arg Val Leu Ala Thr Val Tyr Asn Gly Lys Thr
645 650 655
Thr Tyr Gly Glu Thr Thr Ala Arg Arg Gly Asp Thr Ala Ala Leu Ala
660 665 670
Gln Arg Leu Ser Gly Arg Leu Pro Thr Ser Phe Asn Tyr Gly Ala Val
675 680 685
Lys Ala Glu Thr Ile Thr Glu Leu Leu Ile Arg Met Lys Arg Ala Glu
690 695 700
Thr Tyr Cys Pro Arg Pro Leu Leu Ala Leu Asp Thr Thr Gln Asp Arg
705 710 715 720
Arg Lys Gln Lys Ile Ile Ala Pro Gly Lys Gln Met Val Asn Phe Asp
725 730 735
Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro Phe Phe
740 745 750
Phe Ser Asp Val Arg Ser Asn Phe Ser Lys Leu Val Glu Thr Ile Asn
755 760 765
Gln Met Gln Glu Asp Met Ser Glu Phe Lys Lys Pro Val Ala Leu Lys
770 775 780
Val Lys Ala Lys Asn Leu Ile Val Thr Glu Ser Gly Ala Pro Pro Thr
785 790 795 800
Asp Leu Gln Lys Met Val Met Gly Asn Thr Lys Pro Val Glu Leu Ile
805 810 815
Leu Asp Gly Lys Thr Val Ala Ile Cys Arg Ala Thr Gly Val Phe Gly
820 825 830
Thr Ala Tyr Leu Val Pro Arg His Leu Phe Ala Glu Lys Tyr Asp Glu
835 840 845
Ile Met Leu Asp Gly Arg Ala Met Thr Asp Ser Asp Tyr Arg Val Phe
850 855 860
Glu Phe Glu Ile Lys Val Lys Gly Gln Asp Met Leu Ser Asp Ala Ala
865 870 875 880
Leu Met Val Leu His Arg Gly Asn Arg Val Arg Asp Ile Thr Lys His
885 890 895
Phe Arg Asp Val Ala Arg Met Gln Lys Gly Thr Pro Val Val Gly Val
900 905 910
Ile Asn Asn Ala Asp Val Gly Arg Leu Ile Phe Ser Gly Glu Ala Leu
915 920 925
Thr Tyr Lys Asp Ile Val Val Cys Met Asp Gly Asp Thr Met Pro Gly
930 935 940
Leu Phe Ala Tyr Lys Ala Ala Thr Lys Ala Gly Tyr Cys Gly Gly Ala
945 950 955 960
Val Leu Ala Lys Asp Gly Ala Glu Thr Phe Ile Val Gly Thr His Ser
965 970 975
Ala Gly Gly Asn Gly Val Gly Tyr Cys Ser Cys Val Ser Arg Ser Met
980 985 990
Leu Leu Lys Met Lys Ala His Ile Asp Pro Glu Pro His His Glu
995 1000 1005
<210>3
<211>6990
<212>DNA
<213>Foot-and-mouth disease virus
<220>
<221>CDS
<222>(1)..(6990)
<223>
<400>3
atg agc acg act gat tgt ttt atc gct ttg ttg tac gct ttt aga gag 48
Met Ser Thr Thr Asp Cys Phe Ile Ala Leu Leu Tyr Ala Phe Arg Glu
1 5 10 15
atc aaa aca ctg ttc tta tca cgt gca caa ggc aag atg gaa ttc aca 96
Ile Lys Thr Leu Phe Leu Ser Arg Ala Gln Gly Lys Met Glu Phe Thr
20 25 30
ctt cac aac ggt gag aag aaa aca ttc tac tcc agg ccc aat aac cac 144
Leu His Asn Gly Glu Lys Lys Thr Phe Tyr Ser Arg Pro Asn Asn His
35 40 45
gac aac tgc tgg ttg aac acc atc ctc cag ttg ttt agg tac gtc gat 192
Asp Asn Cys Trp Leu Asn Thr Ile Leu Gln Leu Phe Arg Tyr Val Asp
50 55 60
gaa cct ttc ttc gac tgg gtc tac aac tcc ccc gag aat ctc aca ctt 240
Glu Pro Phe Phe Asp Trp Val Tyr Asn Ser Pro Glu Asn Leu Thr Leu
65 70 75 80
gat gcc atc aaa caa ctg gaa gaa att act ggt ctt gag ctg cac gag 288
Asp Ala Ile Lys Gln Leu Glu Glu Ile Thr Gly Leu Glu Leu His Glu
85 90 95
ggt gga cca ccc gct ctc gtt att tgg aac atc aaa cac ctg ctc agc 336
Gly Gly Pro Pro Ala Leu Val Ile Trp Asn Ile Lys His Leu Leu Ser
100 105 110
acc gga atc ggc acc gct tcg cga ccc agc gaa gtg tgc atg gta gac 384
Thr Gly Ile Gly Thr Ala Ser Arg Pro Ser Glu Val Cys Met Val Asp
115 120 125
ggg acg gac atg tgt ttg gct gac ttc cat gct ggc att ttc ctg aaa 432
Gly Thr Asp Met Cys Leu Ala Asp Phe His Ala Gly Ile Phe Leu Lys
130 135 140
gga cag gaa cac gct gtg ttc gcc tgc gtc acc tcc aat ggg tgg tac 480
Gly Gln Glu His Ala Val Phe Ala Cys Val Thr Ser Asn Gly Trp Tyr
145 150 155 160
gcg att gac gac gaa gac ttt tac ccc tgg acg ccg gac ccg tcc gac 528
Ala Ile Asp Asp Glu Asp Phe Tyr Pro Trp Thr Pro Asp Pro Ser Asp
165 170 175
gtt ctg gtg ttt gtc ccg tac gat caa gag cca ctc aac gga gaa tgg 576
Val Leu Val Phe Val Pro Tyr Asp Gln Glu Pro Leu Asn Gly Glu Trp
180 185 190
aaa gca aag gtc cag aga cgg ctc aga gga gcc ggg caa tcc agc ccg 624
Lys Ala Lys Val Gln Arg Arg Leu Arg Gly Ala Gly Gln Ser Ser Pro
195 200 205
gcg act ggg tca cag aac cag tca ggc aac act gga agc atc atc aac 672
Ala Thr Gly Ser Gln Asn Gln Ser Gly Asn Thr Gly Ser Ile Ile Asn
210 215 220
aac tac tac atg cag caa tac cag aac tcc atg gac aca caa ctt gga 720
Asn Tyr Tyr Met Gln Gln Tyr Gln Asn Ser Met Asp Thr Gln Leu Gly
225 230 235 240
gac aac gct att agc gga ggc tcc aac gaa ggt tcc act gac acc acc 768
Asp Asn Ala Ile Ser Gly Gly Ser Asn Glu Gly Ser Thr Asp Thr Thr
245 250 255
tcc aca cac aca aac act acc caa aac aac gat tgg ttc tcg cgc ctg 816
Ser Thr His Thr Asn Thr Thr Gln Asn Asn Asp Trp Phe Ser Arg Leu
260 265 270
gct agt tcc gcc ttc agc gga ctg ttc ggc gcg ctt ctg gct gac aag 864
Ala Ser Ser Ala Phe Ser Gly Leu Phe Gly Ala Leu Leu Ala Asp Lys
275 280 285
aaa acg gaa gaa aca act ttg ctt gaa gac cgc atc ctt acc acc agg 912
Lys Thr Glu Glu Thr Thr Leu Leu Glu Asp Arg Ile Leu Thr Thr Arg
290 295 300
aac ggc cac acg acg tcg acg acc cag tcg agc gtt ggc gtg aca tac 960
Asn Gly His Thr Thr Ser Thr Thr Gln Ser Ser Val Gly Val Thr Tyr
305 310 315 320
ggt tac gct gtg gct gag gac gcg gta tca gga cct aac acc tca ggt 1008
Gly Tyr Ala Val Ala Glu Asp Ala Val Ser Gly Pro Asn Thr Ser Gly
325 330 335
ctc gag acc cgt gtt caa caa gcg gag cgg ttc ttc aaa aag cac ttg 1056
Leu Glu Thr Arg Val Gln Gln Ala Glu Arg Phe Phe Lys Lys His Leu
340 345 350
ttt gat tgg aca ccg aat ttg gct ttt gga tac tgt cac tac ctg gaa 1104
Phe Asp Trp Thr Pro Asn Leu Ala Phe Gly Tyr Cys His Tyr Leu Glu
355 360 365
ctc ccc act gag cac aaa ggt gtg tat ggc agt ctc atg gac tcg tac 1152
Leu Pro Thr Glu His Lys Gly Val Tyr Gly Ser Leu Met Asp Ser Tyr
370 375 380
gcc tac atg aga aac ggg tgg gac ata gag gtg act gct gtt gga aac 1200
Ala Tyr Met Arg Asn Gly Trp Asp Ile Glu Val Thr Ala Val Gly Asn
385 390 395 400
cag ttc aac ggc ggt tgt ctc ctt gtt gca ctt gtg cca gag ctg aag 1248
Gln Phe Asn Gly Gly Cys Leu Leu Val Ala Leu Val Pro Glu Leu Lys
405 410 415
agc ctt gac act cgg cag aaa tac caa ctg acc ctt ttc ccc cat cag 1296
Ser Leu Asp Thr Arg Gln Lys Tyr Gln Leu Thr Leu Phe Pro His Gln
420 425 430
ttc atc aac cca cgc acc aac atg acg gcc cac atc aac gtg ccc ttt 1344
Phe Ile Asn Pro Arg Thr Asn Met Thr Ala His Ile Asn Val Pro Phe
435 440 445
ata ggt gtt aac agg tat gac cag tac atg ctc cac aaa ccg tgg acg 1392
Ile Gly Val Asn Arg Tyr Asp Gln Tyr Met Leu His Lys Pro Trp Thr
450 455 460
ctt gtt gtg atg gtg gtg gcc cca ctc act gtc aag act ggt ggc tct 1440
Leu Val Val Met Val Val Ala Pro Leu Thr Val Lys Thr Gly Gly Ser
465 470 475 480
gaa cag atc aag gtc tac atg aat gca gca ccg acc tac gta cac gtg 1488
Glu Gln Ile Lys Val Tyr Met Asn Ala Ala Pro Thr Tyr Val His Val
485 490 495
gca ggg gag ctc ccc tcg aaa gag ggg ata gtt ccc gtt gcg tgt gcg 1536
Ala Gly Glu Leu Pro Ser Lys Glu Gly Ile Val Pro Val Ala Cys Ala
500 505 510
gac ggt tac ggt aac atg gtg acc acg gac ccg aaa act gcc gac cca 1584
Asp Gly Tyr Gly Asn Met Val Thr Thr Asp Pro Lys Thr Ala Asp Pro
515 520 525
gtg tac ggg aaa gtg ttc aac ccc ccc cgg acg aat ctc ccc ggg cgc 1632
Val Tyr Gly Lys Val Phe Asn Pro Pro Arg Thr Asn Leu Pro Gly Arg
530 535 540
ttc aca aac ttc ctt gat gtc gcg gag gcg tgt cca acc ttc ctc cgc 1680
Phe Thr Asn Phe Leu Asp Val Ala Glu Ala Cys Pro Thr Phe Leu Arg
545 550 555 560
ttc gga gaa gta cca ttt gtg aag acg gtg aac tct ggt gac cgt ttg 1728
Phe Gly Glu Val Pro Phe Val Lys Thr Val Asn Ser Gly Asp Arg Leu
565 570 575
cta gcc aag ttt gat gtc tcg ctc gct gcg ggc cat atg tcc aac acc 1776
Leu Ala Lys Phe Asp Val Ser Leu Ala Ala Gly His Met Ser Asn Thr
580 585 590
tac ttg gct ggt ctg gcg cag tac tac aca cag tac agt ggt acc atg 1824
Tyr Leu Ala Gly Leu Ala Gln Tyr Tyr Thr Gln Tyr Ser Gly Thr Met
595 600 605
aat gtt cac ttc atg ttc acc ggg ccc acg gat gcc aag gcc cgg tac 1872
Asn Val His Phe Met Phe Thr Gly Pro Thr Asp Ala Lys Ala Arg Tyr
610 615 620
atg gtg gcc tac att cct ccc ggc atg aca ccg ccc aca gac cct gag 1920
Met Val Ala Tyr Ile Pro Pro Gly Met Thr Pro Pro Thr Asp Pro Glu
625 630 635 640
cgc gcc gcc cac tgt atc cac tcc gag tgg gac act ggt cta aac tcc 1968
Arg Ala Ala His Cys Ile His Ser Glu Trp Asp Thr Gly Leu Asn Ser
645 650 655
aaa ttc acc ttt tcc ata ccc tac ctc tct gct gct gac tac gca tac 2016
Lys Phe Thr Phe Ser Ile Pro Tyr Leu Ser Ala Ala Asp Tyr Ala Tyr
660 665 670
acc gcc tct gac acg gcg gag acc aca agt gtg caa gga tgg gtg tgc 2064
Thr Ala Ser Asp Thr Ala Glu Thr Thr Ser Val Gln Gly Trp Val Cys
675 680 685
atc tac cag atc acc cac ggc aag gct gaa gga gac gca ctg gtc gtt 2112
Ile Tyr Gln Ile Thr His Gly Lys Ala Glu Gly Asp Ala Leu Val Val
690 695 700
tct gtc agc gcc ggc aaa gac ttt gag ttt cgc ctg ccc gtt gac gcg 2160
Ser Val Ser Ala Gly Lys Asp Phe Glu Phe Arg Leu Pro Val Asp Ala
705 710 715 720
cgc cgg caa acc acc act acc ggc gag tca gca gac ccg gta aca acc 2208
Arg Arg Gln Thr Thr Thr Thr Gly Glu Ser Ala Asp Pro Val Thr Thr
725 730 735
acg gtc gag aac tac gga gga gaa act cag aca gcc agg cgg ctt cac 2256
Thr Val Glu Asn Tyr Gly Gly Glu Thr Gln Thr Ala Arg Arg Leu His
740 745 750
act gat gtt gcc ttc gtt ctt gac agg ttt gtg aaa ctc act gca ccc 2304
Thr Asp Val Ala Phe Val Leu Asp Arg Phe Val Lys Leu Thr Ala Pro
755 760 765
aag aac att cag acc ctt gac ctc atg caa att ccc tca cac acg ctg 2352
Lys Asn Ile Gln Thr Leu Asp Leu Met Gln Ile Pro Ser His Thr Leu
770 775 780
gtt gga gca ctg ctg cgg tct gcg acg tac tac ttc tca gac ctg gag 2400
Val Gly Ala Leu Leu Arg Ser Ala Thr Tyr Tyr Phe Ser Asp Leu Glu
785 790 795 800
gtt gcg att gtc cac aca ggc ccg atc acc tgg gtg ccc aac ggc tcg 2448
Val Ala Ile Val His Thr Gly Pro Ile Thr Trp Val Pro Asn Gly Ser
805 810 815
ccc aag gat gct cta gac aac cag acc aac cca act gcc tac cag aag 2496
Pro Lys Asp Ala Leu Asp Asn Gln Thr Asn Pro Thr Ala Tyr Gln Lys
820 825 830
caa cct gtc acc cgc ttg gcg ctc ccc tac acc gcc ccc cat cgt gtg 2544
Gln Pro Val Thr Arg Leu Ala Leu Pro Tyr Thr Ala Pro His Arg Val
835 840 845
ctg gcg aca gtg tac aac ggg aag acg acg tac ggg gaa aca acc gcg 2592
Leu Ala Thr Val Tyr Asn Gly Lys Thr Thr Tyr Gly Glu Thr Thr Ala
850 855 860
cgg cgt ggc gat acg gcg gcc ctt gca caa aga ctg agt ggg cgg ctg 2640
Arg Arg Gly Asp Thr Ala Ala Leu Ala Gln Arg Leu Ser Gly Arg Leu
865 870 875 880
ccc acc tca ttt aac tac ggc gct gta aag gct gaa acc atc act gag 2688
Pro Thr Ser Phe Asn Tyr Gly Ala Val Lys Ala Glu Thr Ile Thr Glu
885 890 895
ctt ttg att cgc atg aaa cgt gcg gag aca tac tgc cct agg cct ttg 2736
Leu Leu Ile Arg Met Lys Arg Ala Glu Thr Tyr Cys Pro Arg Pro Leu
900 905 910
cta gct ctt gac acc act cag gac cgc cgt aaa cag aag atc att gca 2784
Leu Ala Leu Asp Thr Thr Gln Asp Arg Arg Lys Gln Lys Ile Ile Ala
915 920 925
cct gga aag cag atg gtg aac ttt gac ctg ctc aag ttg gca gga gac 2832
Pro Gly Lys Gln Met Val Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp
930 935 940
gtc gag tcc aac cct ggg ccc ttc ttc ttc tcc gac gtc agg tcg aat 2880
Val Glu Ser Asn Pro Gly Pro Phe Phe Phe Ser Asp Val Arg Ser Asn
945 950 955 960
ttt tcc aag ctg gtt gaa acc atc aac cag atg cag gag gac atg tca 2928
Phe Ser Lys Leu Val Glu Thr Ile Asn Gln Met Gln Glu Asp Met Ser
965 970 975
aca aaa cac gga ccc gac ttt aac cgg ttg gtg tct gca ttt gag gaa 2976
Thr Lys His Gly Pro Asp Phe Asn Arg Leu Val Ser Ala Phe Glu Glu
980 985 990
ctg gcc act gga gtg aag gct atc agg acc ggc ctc gac gag gcc aaa 3024
Leu Ala Thr Gly Val Lys Ala Ile Arg Thr Gly Leu Asp Glu Ala Lys
995 1000 1005
ccc tgg tac aag ctc atc aag ctc cta agc cgc ctg tca tgc atg 3069
Pro Trp Tyr Lys Leu Ile Lys Leu Leu Ser Arg Leu Ser Cys Met
1010 1015 1020
gcc gct gta gca gca cgg tca aag gac cca gtc ctt gtg gcc atc 3114
A1a Ala Val Ala Ala Arg Ser Lys Asp Pro Val Leu Val Ala Ile
1025 1030 1035
atg ctg gct gac acc ggc ctt gag att ctg gac agc acc ttt gtc 3159
Met Leu Ala Asp Thr Gly Leu Glu Ile Leu Asp Ser Thr Phe Val
1040 1045 1050
gtg aag aag atc tcc gac tcg ctc tcc agt ctc ttt cac gtg cca 3204
Val Lys Lys Ile Ser Asp Ser Leu Ser Ser Leu Phe His Val Pro
1055 1060 1065
gcc ccc gtc ttc agt ttc gga gct ccg atc ctg ttg gcc ggg ttg 3249
Ala Pro Val Phe Ser Phe Gly Ala Pro Ile Leu Leu Ala Gly Leu
1070 1075 1080
gtc aaa gtc gcc tcg agt ttc ttc cgg tcc aca ccc gaa gac ctt 3294
Val Lys Val Ala Ser Ser Phe Phe Arg Ser Thr Pro Glu Asp Leu
1085 1090 1095
gag aga gcg gag aaa cag ctc aaa gca cgt gac atc aat gac ata 3339
Glu Arg Ala Glu Lys Gln Leu Lys Ala Arg Asp Ile Asn Asp Ile
1100 1105 1110
ttc gcc att ctc aag aac ggc gag tgg ttg gtc aaa ctg att ctt 3384
Phe Ala Ile Leu Lys Asn Gly Glu Trp Leu Val Lys Leu Ile Leu
1115 1120 1125
gcc atc cgc gac tgg atc aag gca tgg atc gcc tca gaa gaa aag 3429
Ala Ile Arg Asp Trp Ile Lys Ala Trp Ile Ala Ser Glu Glu Lys
1130 1135 1140
ttt gtc acc atg aca gac ctg gtg cct ggc atc ctt gaa aag cag 3474
Phe Val Thr Met Thr Asp Leu Val Pro Gly Ile Leu Glu Lys Gln
1145 1150 1155
cgg gac ctc aac gac cca agc aag tac aag gag gcc aag gag tgg 3519
Arg Asp Leu Asn Asp Pro Ser Lys Tyr Lys Glu Ala Lys Glu Trp
1160 1165 1170
cta gac aac gcg cgc caa gcg tgt ttg aag agc ggg aac acc cac 3564
Leu Asp Asn Ala Arg Gln Ala Cys Leu Lys Ser Gly Asn Thr His
1175 1180 1185
atc gca aac ctt tgc aaa gtg gtt gcc cca gca ccc agc agg tcg 3609
Ile Ala Asn Leu Cys Lys Val Val Ala Pro Ala Pro Ser Arg Ser
1190 1195 1200
agg ccc gaa ccc gtg gtc gtt tgc ctc cgt ggc aaa tcg ggc cag 3654
Arg Pro Glu Pro Val Val Val Cys Leu Arg Gly Lys Ser Gly Gln
1205 1210 1215
ggt aag agc ttc ctt gcg aac gtg ctt gca caa gca att tca acc 3699
Gly Lys Ser Phe Leu Ala Asn Val Leu Ala Gln Ala Ile Ser Thr
1220 1225 1230
cac ttc act ggc aga act gat tca gtt tgg tac tgc cca cct gac 3744
His Phe Thr Gly Arg Thr Asp Ser Val Trp Tyr Cys Pro Pro Asp
1235 1240 1245
cct gat cac ttc gac ggt tac aac caa cag acc gtt gta gtg atg 3789
Pro Asp His Phe Asp Gly Tyr Asn Gln Gln Thr Val Val Val Met
1250 1255 1260
gat gat ttg ggc cag aac ccc gac ggg aag gac ttc aag tac ttc 3834
Asp Asp Leu Gly Gln Asn Pro Asp Gly Lys Asp Phe Lys Tyr Phe
1265 1270 1275
gcc caa atg gtt tca acc acg ggg ttc atc ccg ccc atg gct tca 3879
Ala Gln Met Val Ser Thr Thr Gly Phe Ile Pro Pro Met Ala Ser
1280 1285 1290
ctc gaa gac aaa ggc aaa cct ttc aac agc aag gtc atc atc gcc 3924
Leu Glu Asp Lys Gly Lys Pro Phe Asn Ser Lys Val Ile Ile Ala
1295 1300 1305
acc acc aac ctg tac tcg ggt ttc acc ccg aga act atg gtg tgc 3969
Thr Thr Asn Leu Tyr Ser Gly Phe Thr Pro Arg Thr Met Val Cys
1310 1315 1320
cct gat gca ctg aac cga agg ttc cac ttc gac att gac gtg agc 4014
Pro Asp Ala Leu Asn Arg Arg Phe His Phe Asp Ile Asp Val Ser
1325 1330 1335
gcc aag gac ggg tat aaa att aac aac aaa ttg gac att acc aaa 4059
Ala Lys Asp Gly Tyr Lys Ile Asn Asn Lys Leu Asp Ile Thr Lys
1340 1345 1350
gct ctt gaa gac acc cac acc aac cct gtg gca atg ttt aaa tac 4104
Ala Leu Glu Asp Thr His Thr Asn Pro Val Ala Met Phe Lys Tyr
1355 1360 1365
gac tgt gcc ctt ctc aac ggc atg gcc gtc gaa atg aag aga atg 4149
Asp Cys Ala Leu Leu Asn Gly Met Ala Val Glu Met Lys Arg Met
1370 1375 1380
caa caa gac atg ttc aag cct caa ccg ccc ctc cag aac gtc tac 4194
Gln Gln Asp Met Phe Lys Pro Gln Pro Pro Leu Gln Asn Val Tyr
1385 1390 1395
cag ctt gtt cag gag gtg att gac cgg gtc gag ctc cac gag aag 4239
Gln Leu Val Gln Glu Val Ile Asp Arg Val Glu Leu His Glu Lys
1400 1405 1410
gtg tcg agc cac ccg att ttt aaa cag atc tct att cct tcc caa 4284
Val Ser Ser His Pro Ile Phe Lys Gln Ile Ser Ile Pro Ser Gln
1415 1420 1425
aag tct gtg ctg tac ttt ctc att gag aag ggc cag cac gaa gca 4329
Lys Ser Val Leu Tyr Phe Leu Ile Glu Lys Gly Gln His Glu Ala
1430 1435 1440
gca att gaa ttc ttt gag ggg atg gtg cac gac tcc atc aag gag 4374
Ala Ile Glu Phe Phe Glu Gly Met Val His Asp Ser Ile Lys Glu
1445 1450 1455
gag ctc cgg cct ctc att caa cag acc tca ttt gtg aag cgc gct 4419
Glu Leu Arg Pro Leu Ile Gln Gln Thr Ser Phe Val Lys Arg Ala
1460 1465 1470
ttc aag cgc ctg aag gaa aac ttt gag ata gtt gcc ctg tgt ttg 4464
Phe Lys Arg Leu Lys Glu Asn Phe Glu Ile Val Ala Leu Cys Leu
1475 1480 1485
act ctt ttg gca aac ata gtg atc atg atc cgc gag act cgc aag 4509
Thr Leu Leu Ala Asn Ile Val Ile Met Ile Arg Glu Thr Arg Lys
1490 1495 1500
agg cag caa atg gtg gat gat gca gtg aat gag tac att gag aag 4554
Arg Gln Gln Met Val Asp Asp Ala Val Asn Glu Tyr Ile Glu Lys
1505 1510 1515
gca aac atc acc acg gat gac aag act ctt gac gag gcg gaa aag 4599
Ala Asn Ile Thr Thr Asp Asp Lys Thr Leu Asp Glu Ala Glu Lys
1520 1525 1530
aac cct ttg gaa acc agc ggt gcc acc act gtt ggt ttc aga gag 4644
Asn Pro Leu Glu Thr Ser Gly Ala Thr Thr Val Gly Phe Arg Glu
1535 1540 1545
aaa act ctc ccg ggg cac aag gcg agt gat gac gtg aac tcc gag 4689
Lys Thr Leu Pro Gly His Lys Ala Ser Asp Asp Val Asn Ser Glu
1550 1555 1560
ccc gcc aaa tcc gtg gaa gaa caa cca caa gct gaa gga ccc tac 4734
Pro Ala Lys Ser Val Glu Glu Gln Pro Gln Ala Glu Gly Pro Tyr
1565 1570 1575
gcc gga cca ctc gag cgt cag aaa cct ctg aaa gtg aga gcc aag 4779
Ala Gly Pro Leu Glu Arg Gln Lys Pro Leu Lys Val Arg Ala Lys
1580 1585 1590
ctc ccg cag cag gag ggg ccc tac gct ggt ccg atg gag aga cag 4824
Leu Pro Gln Gln Glu Gly Pro Tyr Ala Gly Pro Met Glu Arg Gln
1595 1600 1605
aaa ccg ctg aaa gtg aaa gca aaa gcc ccg gtc gtt aag gaa gga 4869
Lys Pro Leu Lys Val Lys Ala Lys Ala Pro Val Val Lys Glu Gly
1610 1615 1620
cct tac gaa gga ccg gtc aag aaa cct gtc gct ttg aaa gtg aaa 4914
Pro Tyr Glu Gly Pro Val Lys Lys Pro Val Ala Leu Lys Val Lys
1625 1630 1635
gca aag aac ttg att gtc act gag agt ggt gct ccc ccg act gac 4959
Ala Lys Asn Leu Ile Val Thr Glu Ser Gly Ala Pro Pro Thr Asp
1640 1645 1650
ttg caa aag atg gtc atg ggt aac acc aag cct gtt gag ctc atc 5004
Leu Gln Lys Met Val Met Gly Asn Thr Lys Pro Val Glu Leu Ile
1655 1660 1665
ctc gac ggg aag acg gtg gcc atc tgc cgc gcc acc gga gtg ttt 5049
Leu Asp Gly Lys Thr Val Ala Ile Cys Arg Ala Thr Gly Val Phe
1670 1675 1680
ggt act gcc tac ctt gtt cct cgt cat ctc ttc gca gag aag tat 5094
Gly Thr Ala Tyr Leu Val Pro Arg His Leu Phe Ala Glu Lys Tyr
1685 1690 1695
gac gag atc atg ctg gac ggc aga gcc atg aca gac agt gac tac 5139
Asp Glu Ile Met Leu Asp Gly Arg Ala Met Thr Asp Ser Asp Tyr
1700 1705 1710
aga gtg ttt gag ttt gag att aaa gta aaa gga cag gac atg ctc 5184
Arg Val Phe Glu Phe Glu Ile Lys Val Lys Gly Gln Asp Met Leu
1715 1720 1725
tca gac gcc gcg ctc atg gtg ctc cac cgt ggg aat cgc gtg cgg 5229
Ser Asp Ala Ala Leu Met Val Leu His Arg Gly Asn Arg Val Arg
1730 1735 1740
gac atc acg aag cac ttc cgt gat gtg gca aga atg cag aaa ggc 5274
Asp Ile Thr Lys His Phe Arg Asp Val Ala Arg Met Gln Lys Gly
1745 1750 1755
acc ccc gtc gtc ggc gta atc aac aac gct gat gtt ggg aga ctg 5319
Thr Pro Val Val Gly Val Ile Asn Asn Ala Asp Val Gly Arg Leu
1760 1765 1770
att ttc tct ggt gag gcc ctt acc tac aag gac att gta gtg tgc 5364
Ile Phe Ser Gly Glu Ala Leu Thr Tyr Lys Asp Ile Val Val Cys
1775 1780 1785
atg gac gga gac acc atg ccc ggc ctc ttc gcc tac aaa gct gcc 5409
Met Asp Gly Asp Thr Met Pro Gly Leu Phe Ala Tyr Lys Ala Ala
1790 1795 1800
acc aag gca ggt tac tgt gga gga gcc gtt ctt gca aag gac gga 5454
Thr Lys Ala Gly Tyr Cys Gly Gly Ala Val Leu Ala Lys Asp Gly
1805 1810 1815
gcc gag act ttc atc gtt ggc act cac tct gca ggt ggc aat gga 5499
Ala Glu Thr Phe Ile Val Gly Thr His Ser Ala Gly Gly Asn Gly
1820 1825 1830
gtt gga tac tgc tca tgc gtt tcc agg tct atg ctg ctc aaa atg 5544
Val Gly Tyr Cys Ser Cys Val Ser Arg Ser Met Leu Leu Lys Met
1835 1840 1845
aag gca cac atc gat ccc gaa cca cac cac gag gga ttg ata gtt 5589
Lys Ala His Ile Asp Pro Glu Pro His His Glu Gly Leu Ile Val
1850 1855 1860
gac acc aga gat gtt gag gag cgc gtg cat gtc atg cgc aaa acc 5634
Asp Thr Arg Asp Val Glu Glu Arg Val His Val Met Arg Lys Thr
1865 1870 1875
aag ctc gca ccc act gtg gca cac ggt gtg ttt aac cct gaa ttc 5679
Lys Leu Ala Pro Thr Val Ala His Gly Val Phe Asn Pro Glu Phe
1880 1885 1890
ggg cct gcc gcc ttg tcc aac aaa gac ccg cgc ctg gat gaa gga 5724
Gly Pro Ala Ala Leu Ser Asn Lys Asp Pro Arg Leu Asp Glu Gly
1895 1900 1905
gtt gtc ctc gat gaa gcc atc ttc tcc aaa cac aag gga gac aca 5769
Val Val Leu Asp Glu Ala Ile Phe Ser Lys His Lys Gly Asp Thr
1910 1915 1920
aag atg tct gag gag gac aaa gcg ctg ttc cgc cgc tgc gct gcc 5814
Lys Met Ser Glu Glu Asp Lys Ala Leu Phe Arg Arg Cys Ala Ala
1925 1930 1935
gac tac gcg tcg cgt ctg cac agc gtg ctg ggt acg gca aac gcc 5859
Asp Tyr Ala Ser Arg Leu His Ser Val Leu Gly Thr Ala Asn Ala
1940 1945 1950
cca ctg agc atc tac gag gca att aag ggc gtc gac gga ctt gac 5904
Pro Leu Ser Ile Tyr Glu Ala Ile Lys Gly Val Asp Gly Leu Asp
1955 1960 1965
gcc atg gaa cca gac acc gcg cct ggt ctt ccc tgg gct ctc cag 5949
Ala Met Glu Pro Asp Thr Ala Pro Gly Leu Pro Trp Ala Leu Gln
1970 1975 1980
ggg aaa cgc cgt ggt gcg ctc att gac ttc gag aac ggc act gtc 5994
Gly Lys Arg Arg Gly Ala Leu Ile Asp Phe Glu Asn Gly Thr Val
1985 1990 1995
gga ccc gag gtt aaa gct gcc tta gag ctc atg gag aaa aga gag 6039
Gly Pro Glu Val Lys Ala Ala Leu Glu Leu Met Glu Lys Arg Glu
2000 2005 2010
tac aag ttt gca tgc caa acc ttc ctg aag gac gag att cgc ccg 6084
Tyr Lys Phe Ala Cys Gln Thr Phe Leu Lys Asp Glu Ile Arg Pro
2015 2020 2025
atg gaa aag gta cgt gcc ggc agg act cgc att gtc gac gtc ttg 6129
Met Glu Lys Val Arg Ala Gly Arg Thr Arg Ile Val Asp Val Leu
2030 2035 2040
cct gtt gaa cac att ctt tac acc agg atg atg att ggc aga ttc 6174
Pro Val Glu His Ile Leu Tyr Thr Arg Met Met Ile Gly Arg Phe
2045 2050 2055
tgt gct caa atg cac tca aac aac gga ccg caa att ggc tcg gcg 6219
Cys Ala Gln Met His Ser Asn Asn Gly Pro Gln Ile Gly Ser Ala
2060 2065 2070
gtt ggt tgc aat cct gat gtt gat tgg caa aga ttt ggc aca cac 6264
Val Gly Cys Asn Pro Asp Val Asp Trp Gln Arg Phe Gly Thr His
2075 2080 2085
ttt gct cag tac aga aac gtg tgg gat gtg gac tat tcg gcc ttt 6309
Phe Ala Gln Tyr Arg Asn Val Trp Asp Val Asp Tyr Ser Ala Phe
2090 2095 2100
gat gcc aac cac tgc agt gac gca atg aac atc atg ttt gag gag 6354
Asp Ala Asn His Cys Ser Asp Ala Met Asn Ile Met Phe Glu Glu
2105 2110 2115
gtg ttc aac acg gac ttc ggt ttc cac cca aac gct gag tgg atc 6399
Val Phe Asn Thr Asp Phe Gly Phe His Pro Asn Ala Glu Trp Ile
2120 2125 2130
ctg aaa act ctc gtg aac act gaa cac gcc tat gag aac aaa cgc 6444
Leu Lys Thr Leu Val Asn Thr Glu His Ala Tyr Glu Asn Lys Arg
2135 2140 2145
atc act gtt gag ggc ggg atg cca tct ggt tgt tcc gca aca agc 6489
Ile Thr Val Glu Gly Gly Met Pro Ser Gly Cys Ser Ala Thr Ser
2150 2155 2160
atc atc aac aca att ttg aac aat atc tac gtg ctc tac gcc ttg 6534
Ile Ile Asn Thr Ile Leu Asn Asn Ile Tyr Val Leu Tyr Ala Leu
2165 2170 2175
cgt aga cac tat gag gga gtt gag ctg gac act tac acc atg atc 6579
Arg Arg His Tyr Glu Gly Val Glu Leu Asp Thr Tyr Thr Met Ile
2180 2185 2190
tct tac gga gac gac atc gtg gtt gca agt gat cac gat ctg gac 6624
Ser Tyr Gly Asp Asp Ile Val Val Ala Ser Asp His Asp Leu Asp
2195 2200 2205
ttt gag gcc ctc aag cct cac ttc aaa tcc ctt ggt caa acc atc 6669
Phe Glu Ala Leu Lys Pro His Phe Lys Ser Leu Gly Gln Thr Ile
2210 2215 2220
act cca gct gac aaa agc gac aaa ggt ttt gtt ctt ggt cac tcc 6714
Thr Pro Ala Asp Lys Ser Asp Lys Gly Phe Val Leu Gly His Ser
2225 2230 2235
atc acc gat gtc act ttc ctc aaa aga cac ttt cac atg gat tat 6759
Ile Thr Asp Val Thr Phe Leu Lys Arg His Phe His Met Asp Tyr
2240 2245 2250
gga act ggg ttt tac aaa cct gtg atg gct tcg aag acc ctc gag 6804
Gly Thr Gly Phe Tyr Lys Pro Val Met Ala Ser Lys Thr Leu Glu
2255 2260 2265
gct atc ctc tcc ttt gca cgc cgt ggg acc ata cag gag aag ttg 6849
Ala Ile Leu Ser Phe Ala Arg Arg Gly Thr Ile Gln Glu Lys Leu
2270 2275 2280
atc tcc gtg gca gga ctc gcc gtc cac tct gga cct gac gag tac 6894
Ile Ser Val Ala Gly Leu Ala Val His Ser Gly Pro Asp Glu Tyr
2285 2290 2295
cgg cgt ctc ttt gag cct ttc cag ggc ctc ttt gag att cca agc 6939
Arg Arg Leu Phe Glu Pro Phe Gln Gly Leu Phe Glu Ile Pro Ser
2300 2305 2310
tac aga tca ctt tac ctg cgt tgg gtg aac gcc gtg tgc ggt gac 6984
Tyr Arg Ser Leu Tyr Leu Arg Trp Val Asn Ala Val Cys Gly Asp
2315 2320 2325
gca taa 6990
Ala
<210>4
<211>2329
<212>PRT
<213>Foot-and-mouth disease virus
<400>4
Met Ser Thr Thr Asp Cys Phe Ile Ala Leu Leu Tyr Ala Phe Arg Glu
1 5 10 15
Ile Lys Thr Leu Phe Leu Ser Arg Ala Gln Gly Lys Met Glu Phe Thr
20 25 30
Leu His Asn Gly Glu Lys Lys Thr Phe Tyr Ser Arg Pro Asn Asn His
35 40 45
Asp Asn Cys Trp Leu Asn Thr Ile Leu Gln Leu Phe Arg Tyr Val Asp
50 55 60
Glu Pro Phe Phe Asp Trp Val Tyr Asn Ser Pro Glu Asn Leu Thr Leu
65 70 75 80
Asp Ala Ile Lys Gln Leu Glu Glu Ile Thr Gly Leu Glu Leu His Glu
85 90 95
Gly Gly Pro Pro Ala Leu Val Ile Trp Asn Ile Lys His Leu Leu Ser
100 105 110
Thr Gly Ile Gly Thr Ala Ser Arg Pro Ser Glu Val Cys Met Val Asp
115 120 125
Gly Thr Asp Met Cys Leu Ala Asp Phe His Ala Gly Ile Phe Leu Lys
130 135 140
Gly Gln Glu His Ala Val Phe Ala Cys Val Thr Ser Asn Gly Trp Tyr
145 150 155 160
Ala Ile Asp Asp Glu Asp Phe Tyr Pro Trp Thr Pro Asp Pro Ser Asp
165 170 175
Val Leu Val Phe Val Pro Tyr Asp Gln Glu Pro Leu Asn Gly Glu Trp
180 185 190
Lys Ala Lys Val Gln Arg Arg Leu Arg Gly Ala Gly Gln Ser Ser Pro
195 200 205
Ala Thr Gly Ser Gln Asn Gln Ser Gly Asn Thr Gly Ser Ile Ile Asn
210 215 220
Asn Tyr Tyr Met Gln Gln Tyr Gln Asn Ser Met Asp Thr Gln Leu Gly
225 230 235 240
Asp Asn Ala Ile Ser Gly Gly Ser Asn Glu Gly Ser Thr Asp Thr Thr
245 250 255
Ser Thr His Thr Asn Thr Thr Gln Asn Asn Asp Trp Phe Ser Arg Leu
260 265 270
Ala Ser Ser Ala Phe Ser Gly Leu Phe Gly Ala Leu Leu Ala Asp Lys
275 280 285
Lys Thr Glu Glu Thr Thr Leu Leu Glu Asp Arg Ile Leu Thr Thr Arg
290 295 300
Asn Gly His Thr Thr Ser Thr Thr Gln Ser Ser Val Gly Val Thr Tyr
305 310 315 320
Gly Tyr Ala Val Ala Glu Asp Ala Val Ser Gly Pro Asn Thr Ser Gly
325 330 335
Leu Glu Thr Arg Val Gln Gln Ala Glu Arg Phe Phe Lys Lys His Leu
340 345 350
Phe Asp Trp Thr Pro Asn Leu Ala Phe Gly Tyr Cys His Tyr Leu Glu
355 360 365
Leu Pro Thr Glu His Lys Gly Val Tyr Gly Ser Leu Met Asp Ser Tyr
370 375 380
Ala Tyr Met Arg Asn Gly Trp Asp Ile Glu Val Thr Ala Val Gly Asn
385 390 395 400
Gln Phe Asn Gly Gly Cys Leu Leu Val Ala Leu Val Pro Glu Leu Lys
405 410 415
Ser Leu Asp Thr Arg Gln Lys Tyr Gln Leu Thr Leu Phe Pro His Gln
420 425 430
Phe Ile Asn Pro Arg Thr Asn Met Thr Ala His Ile Asn Val Pro Phe
435 440 445
Ile Gly Val Asn Arg Tyr Asp Gln Tyr Met Leu His Lys Pro Trp Thr
450 455 460
Leu Val Val Met Val Val Ala Pro Leu Thr Val Lys Thr Gly Gly Ser
465 470 475 480
Glu Gln Ile Lys Val Tyr Met Asn Ala Ala Pro Thr Tyr Val His Val
485 490 495
Ala Gly Glu Leu Pro Ser Lys Glu Gly Ile Val Pro Val Ala Cys Ala
500 505 510
Asp Gly Tyr Gly Asn Met Val Thr Thr Asp Pro Lys Thr Ala Asp Pro
515 520 525
Val Tyr Gly Lys Val Phe Asn Pro Pro Arg Thr Asn Leu Pro Gly Arg
530 535 540
Phe Thr Asn Phe Leu Asp Val Ala Glu Ala Cys Pro Thr Phe Leu Arg
545 550 555 560
Phe Gly Glu Val Pro Phe Val Lys Thr Val Asn Ser Gly Asp Arg Leu
565 570 575
Leu Ala Lys Phe Asp Val Ser Leu Ala Ala Gly His Met Ser Asn Thr
580 585 590
Tyr Leu Ala Gly Leu Ala Gln Tyr Tyr Thr Gln Tyr Ser Gly Thr Met
595 600 605
Asn Val His Phe Met Phe Thr Gly Pro Thr Asp Ala Lys Ala Arg Tyr
610 615 620
Met Val Ala Tyr Ile Pro Pro Gly Met Thr Pro Pro Thr Asp Pro Glu
625 630 635 640
Arg Ala Ala His Cys Ile His Ser Glu Trp Asp Thr Gly Leu Asn Ser
645 650 655
Lys Phe Thr Phe Ser Ile Pro Tyr Leu Ser Ala Ala Asp Tyr Ala Tyr
660 665 670
Thr Ala Ser Asp Thr Ala Glu Thr Thr Ser Val Gln Gly Trp Val Cys
675 680 685
Ile Tyr Gln Ile Thr His Gly Lys Ala Glu Gly Asp Ala Leu Val Val
690 695 700
Ser Val Ser Ala Gly Lys Asp Phe Glu Phe Arg Leu Pro Val Asp Ala
705 710 715 720
Arg Arg Gln Thr Thr Thr Thr Gly Glu Ser Ala Asp Pro Val Thr Thr
725 730 735
Thr Val Glu Asn Tyr Gly Gly Glu Thr Gln Thr Ala Arg Arg Leu His
740 745 750
Thr Asp Val Ala Phe Val Leu Asp Arg Phe Val Lys Leu Thr Ala Pro
755 760 765
Lys Asn Ile Gln Thr Leu Asp Leu Met Gln Ile Pro Ser His Thr Leu
770 775 780
Val Gly Ala Leu Leu Arg Ser Ala Thr Tyr Tyr Phe Ser Asp Leu Glu
785 790 795 800
Val Ala Ile Val His Thr Gly Pro Ile Thr Trp Val Pro Asn Gly Ser
805 810 815
Pro Lys Asp Ala Leu Asp Asn Gln Thr Asn Pro Thr Ala Tyr Gln Lys
820 825 830
Gln Pro Val Thr Arg Leu Ala Leu Pro Tyr Thr Ala Pro His Arg Val
835 840 845
Leu Ala Thr Val Tyr Asn Gly Lys Thr Thr Tyr Gly Glu Thr Thr Ala
850 855 860
Arg Arg Gly Asp Thr Ala Ala Leu Ala Gln Arg Leu Ser Gly Arg Leu
865 870 875 880
Pro Thr Ser Phe Asn Tyr Gly Ala Val Lys Ala Glu Thr Ile Thr Glu
885 890 895
Leu Leu Ile Arg Met Lys Arg Ala Glu Thr Tyr Cys Pro Arg Pro Leu
900 905 910
Leu Ala Leu Asp Thr Thr Gln Asp Arg Arg Lys Gln Lys Ile Ile Ala
915 920 925
Pro Gly Lys Gln Met Val Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp
930 935 940
Val Glu Ser Asn Pro Gly Pro Phe Phe Phe Ser Asp Val Arg Ser Asn
945 950 955 960
Phe Ser Lys Leu Val Glu Thr Ile Asn Gln Met Gln Glu Asp Met Ser
965 970 975
Thr Lys His Gly Pro Asp Phe Asn Arg Leu Val Ser Ala Phe Glu Glu
980 985 990
Leu Ala Thr Gly Val Lys Ala Ile Arg Thr Gly Leu Asp Glu Ala Lys
995 1000 1005
Pro Trp Tyr Lys Leu Ile Lys Leu Leu Ser Arg Leu Ser Cys Met
1010 1015 1020
Ala Ala Val Ala Ala Arg Ser Lys Asp Pro Val Leu Val Ala Ile
1025 1030 1035
Met Leu Ala Asp Thr Gly Leu Glu Ile Leu Asp Ser Thr Phe Val
1040 1045 1050
Val Lys Lys Ile Ser Asp Ser Leu Ser Ser Leu Phe His Val Pro
1055 1060 1065
Ala Pro Val Phe Ser Phe Gly Ala Pro Ile Leu Leu Ala Gly Leu
1070 1075 1080
Val Lys Val Ala Ser Ser Phe Phe Arg Ser Thr Pro Glu Asp Leu
1085 1090 1095
Glu Arg Ala Glu Lys Gln Leu Lys Ala Arg Asp Ile Asn Asp Ile
1100 1105 1110
Phe Ala Ile Leu Lys Asn Gly Glu Trp Leu Val Lys Leu Ile Leu
1115 1120 1125
Ala Ile Arg Asp Trp Ile Lys Ala Trp Ile Ala Ser Glu Glu Lys
1130 1135 1140
Phe Val Thr Met Thr Asp Leu Val Pro Gly Ile Leu Glu Lys Gln
1145 1150 1155
Arg Asp Leu Asn Asp Pro Ser Lys Tyr Lys Glu Ala Lys Glu Trp
1160 1165 1170
Leu Asp Asn Ala Arg Gln Ala Cys Leu Lys Ser Gly Asn Thr His
1175 1180 1185
Ile Ala Asn Leu Cys Lys Val Val Ala Pro Ala Pro Ser Arg Ser
1190 1195 1200
Arg Pro Glu Pro Val Val Val Cys Leu Arg Gly Lys Ser Gly Gln
1205 1210 1215
Gly Lys Ser Phe Leu Ala Asn Val Leu Ala Gln Ala Ile Ser Thr
1220 1225 1230
His Phe Thr Gly Arg Thr Asp Ser Val Trp Tyr Cys Pro Pro Asp
1235 1240 1245
Pro Asp His Phe Asp Gly Tyr Asn Gln Gln Thr Val Val Val Met
1250 1255 1260
Asp Asp Leu Gly Gln Asn Pro Asp Gly Lys Asp Phe Lys Tyr Phe
1265 1270 1275
Ala Gln Met Val Ser Thr Thr Gly Phe Ile Pro Pro Met Ala Ser
1280 1285 1290
Leu Glu Asp Lys Gly Lys Pro Phe Asn Ser Lys Val Ile Ile Ala
1295 1300 1305
Thr Thr Asn Leu Tyr Ser Gly Phe Thr Pro Arg Thr Met Val Cys
1310 1315 1320
Pro Asp Ala Leu Asn Arg Arg Phe His Phe Asp Ile Asp Val Ser
1325 1330 1335
Ala Lys Asp Gly Tyr Lys Ile Asn Asn Lys Leu Asp Ile Thr Lys
1340 1345 1350
Ala Leu Glu Asp Thr His Thr Asn Pro Val Ala Met Phe Lys Tyr
1355 1360 1365
Asp Cys Ala Leu Leu Asn Gly Met Ala Val Glu Met Lys Arg Met
1370 1375 1380
Gln Gln Asp Met Phe Lys Pro Gln Pro Pro Leu Gln Asn Val Tyr
1385 1390 1395
Gln Leu Val Gln Glu Val Ile Asp Arg Val Glu Leu His Glu Lys
1400 1405 1410
Val Ser Ser His Pro Ile Phe Lys Gln Ile Ser Ile Pro Ser Gln
1415 1420 1425
Lys Ser Val Leu Tyr Phe Leu Ile Glu Lys Gly Gln His Glu Ala
1430 1435 1440
Ala Ile Glu Phe Phe Glu Gly Met Val His Asp Ser Ile Lys Glu
1445 1450 1455
Glu Leu Arg Pro Leu Ile Gln Gln Thr Ser Phe Val Lys Arg Ala
1460 1465 1470
Phe Lys Arg Leu Lys Glu Asn Phe Glu Ile Val Ala Leu Cys Leu
1475 1480 1485
Thr Leu Leu Ala Asn Ile Val Ile Met Ile Arg Glu Thr Arg Lys
1490 1495 1500
Arg Gln Gln Met Val Asp Asp Ala Val Asn Glu Tyr Ile Glu Lys
1505 1510 1515
Ala Asn Ile Thr Thr Asp Asp Lys Thr Leu Asp Glu Ala Glu Lys
1520 1525 1530
Asn Pro Leu Glu Thr Ser Gly Ala Thr Thr Val Gly Phe Arg Glu
1535 1540 1545
Lys Thr Leu Pro Gly His Lys Ala Ser Asp Asp Val Asn Ser Glu
1550 1555 1560
Pro Ala Lys Ser Val Glu Glu Gln Pro Gln Ala Glu Gly Pro Tyr
1565 1570 1575
Ala Gly Pro Leu Glu Arg Gln Lys Pro Leu Lys Val Arg Ala Lys
1580 1585 1590
Leu Pro Gln Gln Glu Gly Pro Tyr Ala Gly Pro Met Glu Arg Gln
1595 1600 1605
Lys Pro Leu Lys Val Lys Ala Lys Ala Pro Val Val Lys Glu Gly
1610 1615 1620
Pro Tyr Glu Gly Pro Val Lys Lys Pro Val Ala Leu Lys Val Lys
1625 1630 1635
Ala Lys Asn Leu Ile Val Thr Glu Ser Gly Ala Pro Pro Thr Asp
1640 1645 1650
Leu Gln Lys Met Val Met Gly Asn Thr Lys Pro Val Glu Leu Ile
1655 1660 1665
Leu Asp Gly Lys Thr Val Ala Ile Cys Arg Ala Thr Gly Val Phe
1670 1675 1680
Gly Thr Ala Tyr Leu Val Pro Arg His Leu Phe Ala Glu Lys Tyr
1685 1690 1695
Asp Glu Ile Met Leu Asp Gly Arg Ala Met Thr Asp Ser Asp Tyr
1700 1705 1710
Arg Val Phe Glu Phe Glu Ile Lys Val Lys Gly Gln Asp Met Leu
1715 1720 1725
Ser Asp Ala Ala Leu Met Val Leu His Arg Gly Asn Arg Val Arg
1730 1735 1740
Asp Ile Thr Lys His Phe Arg Asp Val Ala Arg Met Gln Lys Gly
1745 1750 1755
Thr Pro Val Val Gly Val Ile Asn Asn Ala Asp Val Gly Arg Leu
1760 1765 1770
Ile Phe Ser Gly Glu Ala Leu Thr Tyr Lys Asp Ile Val Val Cys
1775 1780 1785
Met Asp Gly Asp Thr Met Pro Gly Leu Phe Ala Tyr Lys Ala Ala
1790 1795 1800
Thr Lys Ala Gly Tyr Cys Gly Gly Ala Val Leu Ala Lys Asp Gly
1805 1810 1815
Ala Glu Thr Phe Ile Val Gly Thr His Ser Ala Gly Gly Asn Gly
1820 1825 1830
Val Gly Tyr Cys Ser Cys Val Ser Arg Ser Met Leu Leu Lys Met
1835 1840 1845
Lys Ala His Ile Asp Pro Glu Pro His His Glu Gly Leu Ile Val
1850 1855 1860
Asp Thr Arg Asp Val Glu Glu Arg Val His Val Met Arg Lys Thr
1865 1870 1875
Lys Leu Ala Pro Thr Val Ala His Gly Val Phe Asn Pro Glu Phe
1880 1885 1890
Gly Pro Ala Ala Leu Ser Asn Lys Asp Pro Arg Leu Asp Glu Gly
1895 1900 1905
Val Val Leu Asp Glu Ala Ile Phe Ser Lys His Lys Gly Asp Thr
1910 1915 1920
Lys Met Ser Glu Glu Asp Lys Ala Leu Phe Arg Arg Cys Ala Ala
1925 1930 1935
Asp Tyr Ala Ser Arg Leu His Ser Val Leu Gly Thr Ala Asn Ala
1940 1945 1950
Pro Leu Ser Ile Tyr Glu Ala Ile Lys Gly Val Asp Gly Leu Asp
1955 1960 1965
Ala Met Glu Pro Asp Thr Ala Pro Gly Leu Pro Trp Ala Leu Gln
1970 1975 1980
Gly Lys Arg Arg Gly Ala Leu Ile Asp Phe Glu Asn Gly Thr Val
1985 1990 1995
Gly Pro Glu Val Lys Ala Ala Leu Glu Leu Met Glu Lys Arg Glu
2000 2005 2010
Tyr Lys Phe Ala Cys Gln Thr Phe Leu Lys Asp Glu Ile Arg Pro
2015 2020 2025
Met Glu Lys Val Arg Ala Gly Arg Thr Arg Ile Val Asp Val Leu
2030 2035 2040
Pro Val Glu His Ile Leu Tyr Thr Arg Met Met Ile Gly Arg Phe
2045 2050 2055
Cys Ala Gln Met His Ser Asn Asn Gly Pro Gln Ile Gly Ser Ala
2060 2065 2070
Val Gly Cys Asn Pro Asp Val Asp Trp Gln Arg Phe Gly Thr His
2075 2080 2085
Phe Ala Gln Tyr Arg Asn Val Trp Asp Val Asp Tyr Ser Ala Phe
2090 2095 2100
Asp Ala Asn His Cys Ser Asp Ala Met Asn Ile Met Phe Glu Glu
2105 2110 2115
Val Phe Asn Thr Asp Phe Gly Phe His Pro Asn Ala Glu Trp Ile
2120 2125 2130
Leu Lys Thr Leu Val Asn Thr Glu His Ala Tyr Glu Asn Lys Arg
2135 2140 2145
Ile Thr Val Glu Gly Gly Met Pro Ser Gly Cys Ser Ala Thr Ser
2150 2155 2160
Ile Ile Asn Thr Ile Leu Asn Asn Ile Tyr Val Leu Tyr Ala Leu
2165 2170 2175
Arg Arg His Tyr Glu Gly Val Glu Leu Asp Thr Tyr Thr Met Ile
2180 2185 2190
Ser Tyr Gly Asp Asp Ile Val Val Ala Ser Asp His Asp Leu Asp
2195 2200 2205
Phe Glu Ala Leu Lys Pro His Phe Lys Ser Leu Gly Gln Thr Ile
2210 2215 2220
Thr Pro Ala Asp Lys Ser Asp Lys Gly Phe Val Leu Gly His Ser
2225 2230 2235
Ile Thr Asp Val Thr Phe Leu Lys Arg His Phe His Met Asp Tyr
2240 2245 2250
Gly Thr Gly Phe Tyr Lys Pro Val Met Ala Ser Lys Thr Leu Glu
2255 2260 2265
Ala Ile Leu Ser Phe Ala Arg Arg Gly Thr Ile Gln Glu Lys Leu
2270 2275 2280
Ile Ser Val Ala Gly Leu Ala Val His Ser Gly Pro Asp Glu Tyr
2285 2290 2295
Arg Arg Leu Phe Glu Pro Phe Gln Gly Leu Phe Glu Ile Pro Ser
2300 2305 2310
Tyr Arg Ser Leu Tyr Leu Arg Trp Val Asn Ala Val Cys Gly Asp
2315 2320 2325
Ala
<210>5
<211>636
<212>DNA
<213>Foot-and-mouth disease virus
<220>
<221>CDS
<222>(1)..(633)
<223>
<400>5
acc acc act acc ggc gag tca gca gac ccg gta aca acc acg gtc gag 48
Thr Thr Thr Thr Gly Glu Ser Ala Asp Pro Val Thr Thr Thr Val Glu
1 5 10 15
aac tac gga gga gaa act cag aca gcc agg cgg ctt cac act gat gtt 96
Asn Tyr Gly Gly Glu Thr Gln Thr Ala Arg Arg Leu His Thr Asp Val
20 25 30
gcc ttc gtt ctt gac agg ttt gtg aaa ctc act gca ccc aag aac att 144
Ala Phe Val Leu Asp Arg Phe Val Lys Leu Thr Ala Pro Lys Asn Ile
35 40 45
cag acc ctt gac ctc atg caa att ccc tca cac acg ctg gtt gga gca 192
Gln Thr Leu Asp Leu Met Gln Ile Pro Ser His Thr Leu Val Gly Ala
50 55 60
ctg ctg cgg tct gcg acg tac tac ttc tca gac ctg gag gtt gcg att 240
Leu Leu Arg Ser Ala Thr Tyr Tyr Phe Ser Asp Leu Glu Val Ala Ile
65 70 75 80
gtc cac aca ggc ccg atc acc tgg gtg ccc aac ggc tcg ccc aag gat 288
Val His Thr Gly Pro Ile Thr Trp Val Pro Asn Gly Ser Pro Lys Asp
85 90 95
gct cta gac aac cag acc aac cca act gcc tac cag aag caa cct gtc 336
Ala Leu Asp Asn Gln Thr Asn Pro Thr Ala Tyr Gln Lys Gln Pro Val
100 105 110
acc cgc ttg gcg ctc ccc tac acc gcc ccc cat cgt gtg ctg gcg aca 384
Thr Arg Leu Ala Leu Pro Tyr Thr Ala Pro His Arg Val Leu Ala Thr
115 120 125
gtg tac aac ggg aag acg acg tac ggg gaa aca acc gcg cgg cgt ggc 432
Val Tyr Asn Gly Lys Thr Thr Tyr Gly Glu Thr Thr Ala Arg Arg Gly
130 135 140
gat acg gcg gcc ctt gca caa aga ctg agt ggg cgg ctg ccc acc tca 480
Asp Thr Ala Ala Leu Ala Gln Arg Leu Ser Gly Arg Leu Pro Thr Ser
145 150 155 160
ttt aac tac ggc gct gta aag gct gaa acc atc act gag ctt ttg att 528
Phe Asn Tyr Gly Ala Val Lys Ala Glu Thr Ile Thr Glu Leu Leu Ile
165 170 175
cgc atg aaa cgt gcg gag aca tac tgc cct agg cct ttg cta gct ctt 576
Arg Met Lys Arg Ala Glu Thr Tyr Cys Pro Arg Pro Leu Leu Ala Leu
180 185 190
gac acc act cag gac cgc cgt aaa cag aag atc att gca cct gga aag 624
Asp Thr Thr Gln Asp Arg Arg Lys Gln Lys Ile Ile Ala Pro Gly Lys
195 200 205
cag atg gtg tag 636
Gln Met Val
210
<210>6
<211>211
<212>PRT
<213>Foot-and-mouth disease virus
<400>6
Thr Thr Thr Thr Gly Glu Ser Ala Asp Pro Val Thr Thr Thr Val Glu
1 5 10 15
Asn Tyr Gly Gly Glu Thr Gln Thr Ala Arg Arg Leu His Thr Asp Val
20 25 30
Ala Phe Val Leu Asp Arg Phe Val Lys Leu Thr Ala Pro Lys Asn Ile
35 40 45
Gln Thr Leu Asp Leu Met Gln Ile Pro Ser His Thr Leu Val Gly Ala
50 55 60
Leu Leu Arg Ser Ala Thr Tyr Tyr Phe Ser Asp Leu Glu Val Ala Ile
65 70 75 80
Val His Thr Gly Pro Ile Thr Trp Val Pro Asn Gly Ser Pro Lys Asp
85 90 95
Ala Leu Asp Asn Gln Thr Asn Pro Thr Ala Tyr Gln Lys Gln Pro Val
100 105 110
Thr Arg Leu Ala Leu Pro Tyr Thr Ala Pro His Arg Val Leu Ala Thr
115 120 125
Val Tyr Asn Gly Lys Thr Thr Tyr Gly Glu Thr Thr Ala Arg Arg Gly
130 135 140
Asp Thr Ala Ala Leu Ala Gln Arg Leu Ser Gly Arg Leu Pro Thr Ser
145 150 155 160
Phe Asn Tyr Gly Ala Val Lys Ala Glu Thr Ile Thr Glu Leu Leu Ile
165 170 175
Arg Met Lys Arg Ala Glu Thr Tyr Cys Pro Arg Pro Leu Leu Ala Leu
180 185 190
Asp Thr Thr Gln Asp Arg Arg Lys Gln Lys Ile Ile Ala Pro Gly Lys
195 200 205
Gln Met Val
210

Claims (8)

1. a method for preparing foot-and-mouth disease antigen comprises: the base sequence shown in SEQ ID NO:1, SEQ ID NO:3 or the SEQID NO:5 is cloned into respectively in the baculovirus delivery carrier, makes up and obtain transfer vector; Carry out the DNA reorganization with constructed transfer vector transfection baculovirus, obtain recombinant baculovirus; With the recombinate shape virus infection insect host; Cultivating infected insect host makes it carry out the foot-and-mouth disease antigen expression; Collect and the expressed foot-and-mouth disease antigen of purifying.
2. according to the method for claim 1, it is characterized in that: described baculovirus delivery carrier is selected from AcRP23-lacZ, AcRP6-SC, AcUW1-lacZ, BacPAK6, Bacmid, BlucBacII-pETL, p2Bac, p2Blue, p89B310, pAc360, pAc373, pAcAB3, pAcAB 4, PAcAS3, pAcC129, pAcC4, DZI, pAcGP67, pAcIE1, pAcJP1, pAcMLF2, pAcMLF 7, pAcMLF8, pAcMP1, pAcMP2, pAcRP23, pAcRP25, pAcRW4, pAcsMAG, pAcUW1, pAcUW21, pAcUW2A, pAcUW2B, pAcUW3, pAcUW31, pAcUW41, pAcUW42, pAcUW43, pAcUW51, pAcVC2, pAcVC3, pAcYM1, pAcJcC5, pBac1, pBac2, pBlueBacIII, pBlueBacHis, pEV55, mXIV, pIEINeo, pJVETL, pJVNhe1, pJVP10, pJVrsMAG, pMBac, pP10, pPAK1, pPBac, pSHONEX 1.1, pSYNXIV VI+, pSYNVI+wp, pSYNXIV VI-, pVL1391, pVL1392, pVL 1393, pVL941, pVL 945, and pVL 985, pVTBac, pBM030, pUAC-5 or other similar baculovirus homologous recombination or transposon vector.
3. according to the method for claim 2, it is characterized in that: described baculovirus delivery carrier is pVL1393.
4. according to the method for claim 1, it is characterized in that: constructed transfer vector is pVL1393-P1-2A3C, pVL1393-ORF or pVL1393-VP1.
5. according to the method for claim 1, it is characterized in that: described baculovirus is selected from BmNPV, AcMNPV, ApNPV, BsSNPV, CfMNPV, EoSNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SlMNPV, SeMNPV or TeNPV.
6. according to the method for claim 1, it is characterized in that: described recombinant baculovirus is selected from following any one recombinant virus: (1) recombinant bombyx mori nuclear polyhedrosis virus rBmNPV-ORF, and its microbial preservation number is: CGMCC NO.1980; (2) recombinant bombyx mori nuclear polyhedrosis virus rBmNPV-P1-2A3C, its microbial preservation number is: CGMCC NO.1979; (3) recombinant bombyx mori nuclear polyhedrosis virus rBmNPV-VP1, its microbial preservation number is: CGMCC NO.1975.
7. according to the method for claim 1, it is characterized in that: described insect host comprises silkworm (Bombyxmori), wild silkworm (Bombyx mandarina), Semen Ricini silkworm (Philosamia cynthia ricim), wild silkworm (Dictyoploca japanica), Philosamia cynthia (Philosamia cynthia pryeri), tussah (Antheraea pernyi), yamama (Antheraea yamamai), wild giant silkworm (Antheraea polyphymus), autographa california (Atographa califorica), tea geometrid (Ectropis obliqua), cabbage army worm (Mamestra brassicae), prodenia litura (Spodoptera littoralis), autumn mythimna separata (Spodoptera frugiperda), cabbage looper (Trichoplusia ni), armyworm (Thaumetopoea wilkinsoni), bollworm (Heliothis armigera), U.S. bollworm (Heliothis zea), oriental tobacco budworm (Heliothis assulta), cigarette beetle (Heliothisvirescens), oriental armyworm (Pseudaletia separata) or gypsymoth (Lymantria dispar).
8. according to the method for claim 7, it is characterized in that: described insect host is silkworm larva or pupa.
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CN101121938B (en) * 2007-03-23 2010-10-06 中国农业科学院兰州兽医研究所 Method for preparing foot-and-mouth disease antigen
WO2011054011A2 (en) 2009-11-02 2011-05-05 The Trustees Of The University Of Pennsylvania Foot and mouth disease virus (fmdv) consensus proteins, coding sequences therefor and vaccines made therefrom
CN101812120A (en) * 2010-02-10 2010-08-25 中国检验检疫科学研究院 South Africa II type foot-and-mouth disease epitope polypeptide and screening method thereof
CN103492410A (en) * 2010-03-12 2014-01-01 梅里亚有限公司 Bluetongue virus recombinant vaccines and uses thereof
CN101816297B (en) * 2010-04-30 2011-07-20 中国计量学院 Method for trapping, preventing and controlling adults of ectropis obliqua
CN102311957B (en) * 2010-07-09 2014-03-12 中国农业科学院生物技术研究所 Hydatidovis soluble antigen preparation method and product thereof
CN102533860B (en) * 2012-01-10 2013-07-24 特菲(天津)生物医药科技有限公司 Foot and mouth disease vaccine and preparation method thereof
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US10435695B2 (en) 2016-09-08 2019-10-08 The Government of the United States of America, as represented by the Secretary of Homeland Security Fusion protein comprising Gaussia luciferase, translation interrupter sequence, and interferon amino acid sequences
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